Improved sugar-depleted fruit or vegetable juice and juice-retaining fruit or vegetable derived matter, methods of producing the same and the use thereof to maintain health and to treat and prevent medical ailments

ABSTRACT

The present invention provides a sugar-depleted fruit or vegetable juice product, wherein said juice product is a fruit or vegetable juice or juice-retaining fruit or vegetable derived matter, wherein said juice product contains at least about 5 g/l gluconic acid and said juice product contains any two or three, of (i) at least about 0.5 g/l Ca 2+ , (iii) at least about 1 g/l K + , and (iii) at least about 0.1 g/l Mg2+. Also provided are methods of producing the same and the use thereof to assist in maintaining the health and well-being of a subject and in the treatment and prevention of medical ailments, specifically those associated with the over-consumption of glucose and/or sucrose or inappropriate metabolism of glucose, e.g. metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis.

The present invention provides improved sugar-depleted fruit orvegetable juice and sugar-depleted juice-retaining fruit or vegetablederived matter, methods of producing the same and the use thereof toassist in maintaining the health and well-being of a subject and in thetreatment and prevention of medical ailments, specifically thoseassociated with the over-consumption of glucose and/or sucrose orinappropriate metabolism of glucose, e.g. metabolic syndrome, diabetesmellitus type II, obesity, dyslipidemia, insulin resistance,hypertension and liver steatosis.

For convenience the term “juice product” is used herein to encompassboth the fruit or vegetable juice and juice-retaining fruit or vegetablederived matter to which the invention relates in general. The term“sugar-depleted juice product” is used herein analogously.

The “sugar-depleted juice product” of the invention may be prepared bytreating fruit or vegetable juice or juice-retaining fruit or vegetablederived matter (a “juice product”) with a plurality of enzymes andsupplementing the juice with defined amounts of particular metal ions,thereby resulting in a modified juice product containing reduced amountsof free glucose and optionally sucrose, e.g. substantially oressentially no free glucose or optionally sucrose, but an amount ofgluconic acid corresponding to the amount by which free glucose andoptionally sucrose in the juice product is reduced, e.g. the amount offree glucose and optionally sucrose in the juice product prior to theenzyme treatment, and amounts of said metal ions that ensurepalatability and contribute to the nutritional and therapeutic value ofthe modified juice product. The presence of gluconic acid in combinationwith reduced amounts, preferably the substantial or essential absence,of sucrose and free glucose means the modified juice product of theinvention has a surprisingly advantageous glycaemic profile and thusrenders the modified juice product useful as part of a healthy diet inhealthy subjects, and also surprisingly effective in treating subjectswith or at risk of developing complex metabolic disorders associatedwith the over-consumption of glucose and/or sucrose and/or inappropriatemetabolism of glucose including metabolic syndrome, diabetes, obesity,dyslipidemia, insulin resistance, hypertension and liver steatosis, onaccount of its favourable insulin response and/or favourable effect oninsulin sensitivity.

It is now well appreciated that a diet rich in simple sugars such asglucose and sucrose can lead to health problems, in particular metabolicconditions including diabetes mellitus type II, metabolic syndrome andobesity. It is also well established that the consumption of fruit andvegetables, including the juices thereof, has certain health benefits,potentially including the prevention of certain cancers and heartdisease. However, fruit and vegetable juice products are also richsources of simple sugars, thus diminishing the overall benefit to healthof such foodstuffs. Consequently, it would potentially be advantageousto lower the sugar content of fruit and vegetable juice products if thiscan be done without detrimentally affecting palatability and leaving theother components of the juice product substantially unaffected.

To this end there has been proposed various techniques to reduce thelevels of free glucose in fruit juices.

CH 632137 describes the microbial fermentation of juices to convertsugars to alcohols and the distillation of the alcohols so produced.Procedures such as these radically alter the flavour and nature of thejuice. To address this problem EP 032010 and EP 0554488 describe complexprocedures in which microbes degrade juice sugars without fermentationinto alcohols.

U.S. Pat. No. 3,935,071 describes the conversion of glucose in fruitjuices and other foodstuffs to gluconic acid through the action ofglucose oxidase. The value of such glucose-depleted foodstuffs fordiabetes is mentioned.

An article in A-Magasinet (Norway), Issue 21 September 2007, describes aspeculative approach in which fruit juice is treated with three enzymesto split sucrose into fructose and glucose, to convert fructose toglucose and then to convert glucose to gluconic acid.

Aziz, M. G., et al., 2011, African Journal of Microbiology Research, Vol5(28), 5046-5052, describes the use of glucose-fructose oxidoreductaseand invertase to convert pineapple juice sugars to gluconic acid andsorbitol. Sugar conversion was incomplete and the palatability of theresulting product was not assessed.

It has now been found that simply reducing or removing free glucose fromfruit and vegetable juices results in a less than palatable product thatrequires the introduction of artificial sweeteners and flavourings or aproduct which fails to retain the flavour and mouth-feel of theunmodified juice. These problems are exacerbated when sucrose and/orfree fructose is also reduced/removed from the juice.

It has now been found however that fruit or vegetable juice products inaccordance with the invention can be sugar-depleted, e.g. renderedsubstantially or essentially devoid of glucose and sucrose, by treatmentwith (i) an enzyme which hydrolyses sucrose into glucose and fructose(e.g. invertase) and (ii) an enzyme that converts glucose into gluconicacid (e.g. glucose oxidase) and, upon supplementation with particularcombinations of metal ions, a sugar-depleted juice product with superiorpalatability is obtained which retains sufficiently the taste, flavourand mouth-feel of the unmodified juice product.

As used herein the term “free glucose” refers to glucose that is notcovalently bound to another saccharide molecule, in other words glucosewhich is not part of a di-, oligo- or poly-saccharide, e.g. includingbut not limited to sucrose, amylose, amylopectin, pectin and cellulose.

As used herein, the term “gluconic acid” is generic and represents allthe equilibrium species of gluconic acid in an aqueous medium—e.g.lactone forms (e.g. D-gluconic acid δ-lactone and D-gluconic acidγ-lactone), gluconate salt forms and the acid form. Without wishing tobe bound by theory, it is believed that the supplementation of thesugar-depleted juice product of the invention with the above describedmetal ions causes a favourable shift in the amounts of gluconate-metalsalts present in the sugar-depleted juice product and it is these saltprofiles that promote the retention of the flavour and mouth-feel of theunmodified juice product.

In addition it has been found that the comparatively high gluconic acidcontent of a modified juice in which substantially, e.g. essentially,all of the free glucose and the glucose in sucrose has been converted togluconic acid offers a product with a glycaemic response, a glycaemicindex, a glycaemic load and an insulin response which are significantlylower than those of an untreated juice, i.e. a more favourable glycaemicprofile and lower available carbohydrate content, respectively, which inturn reduces the amount of dietary energy provided by the product.

By way of example, for grape juice, the relative area under theglycaemic curve in response to the consumption of grape juice treated inaccordance with the invention is less than 16% of that of the glycaemiccurve in response to untreated juice. In the case of apple juice, therelative area under the glycaemic curve in response to consumption ofapple juice treated in accordance with the invention is 30% of that ofthe glycaemic curve in response to untreated juice. Other juices woulddisplay corresponding differences in relative area under the glycaemiccurve between modified and unmodified forms, although the exactreductions will depend on the precise content of glucose and othersugars contributing to glycaemic response.

The difference between the insulin response to such a juice product ofthe invention as compared to the insulin response to an unmodified juiceproduct is may be more pronounced than that of the glucose response. Ajuice product of the invention may also increase insulin sensitivity.

Such a modified juice product is therefore surprisingly suited to thetreatment and prevention of complex metabolic disorders associated withthe over-consumption of glucose and/or sucrose or the inappropriatemetabolism of glucose including metabolic syndrome, diabetes, obesity,dyslipidemia, insulin resistance, hypertension and liver steatosis.

Thus, it can be seen that the present invention provides a palatablemodified juice product of superior nutritional value and therapeuticproperties.

Therefore, in a first aspect of the present invention there is provideda sugar-depleted fruit or vegetable juice product, wherein said juiceproduct is a fruit or vegetable juice or juice-retaining fruit orvegetable derived matter, wherein said juice product contains at leastabout 5 g/l gluconic acid and said juice product contains any two orthree, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1 g/l K⁺, and    -   (iii) at least about 0.1 g/l Mg²⁺.

By “juice” it is meant a liquid that has a make-up essentially in directcorrelation to the liquid part of a ripe (or at least human edible)fruit or a vegetable that has been extracted in a method comprising atleast one mechanical means of juice extraction, e.g. by pressing,pulping, mashing, macerating, liquefying and/or sieving. Preferably ajuice in accordance with the invention is the liquid part of a ripefruit or a vegetable that has been extracted in a method comprising atleast one mechanical means of juice extraction, but in other embodimentsit is a concentrated or diluted form thereof. The reference to dilutedforms includes extended juice products, e.g. those referred to as“nectars”, which typically comprise about 50% to about 90%, e.g. about50% to about 80% or about 50% to about 70%, juice. In accordance withthe invention the fruit or vegetable derived constituents of thesediluted or concentrated forms are in direct correlation to the liquidpart of a ripe fruit or a vegetable that has been extracted in a methodcomprising at least one mechanical means of juice extraction becauseessentially the only component added or removed from the extractedliquid is water. The extraction methods described above may alsocomprise enzyme treatments which breakdown biopolymers. Conveniently theterm “juice” can be taken as that defined by the standards of labellingin the EU (no sugar added etc.).

By “juice-retaining fruit or vegetable derived matter” it is meant asubstance substantially, preferably essentially, made up of, e.g.consisting of, fruit or vegetable derived matter wherein substantially,preferably essentially, none of the juice of the fruit or vegetable hasbeen removed. A juice-retaining fruit or vegetable derived matter of theinvention includes purees, pastes, and stews.

By “sugar-depleted” it is meant that a juice product has a reducedamount of free glucose and optionally a reduced amount of sucrose thattogether result in a reduced glycaemic response in a subject as measuredby the area under the curve (AUC) of a subject's blood glucose(preferably capillary blood glucose) levels over time, preferably overabout 15 mins, 30 mins, 45 mins, 60 mins, 75 mins, 90 mins, 105 mins,120 mins, 150 mins, 180 mins, 210 mins or 240 mins immediately followingconsumption of the sugar-depleted juice product, relative to a juiceproduct that has been prepared from the same fruit or vegetable in thesame way but which is not sugar-depleted. Preferably the AUC iscalculated as the incremental AUC (iAUC), i.e. all area below the curvebut above the fasting blood glucose concentration. Preferably the AUC,e.g. the iAUC, is calculated over about 120 mins. The glycaemic responsefor each product should be determine in the same way.

The sugar-depleted juice product preferably results in an area under thecurve as defined above in response to its consumption that is no morethan 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46, 44, 42,40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 5 or 1%of that of a juice product that has been prepared from the same fruit orvegetable in the same way but which is not sugar-depleted.

Expressed alternatively, “sugar depleted” means that a juice product hasa reduced amount of free glucose and optionally a reduced amount ofsucrose that together result in the juice product having a reducedglycaemic load relative to that of a juice product that has beenprepared from the same fruit or vegetable in the same way but which isnot sugar-depleted. The sugar-depleted juice product preferably has aglycaemic load which is no more than 75%, e.g. no more than about 70,65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22,20, 18, 16, 14, 12, 10, 5 or 1%, of that of a juice product that hasbeen prepared from the same fruit or vegetable in the same way but whichis not sugar-depleted. The glycaemic load of each product should bedetermined in the same way.

For the purposes of the invention, the glycaemic load of a foodstuff iscalculated as the amount of available carbohydrate in a standard portionof the foodstuff multiplied by the glycaemic index (GI) of the foodstuffdivided by 100. For the juice product of the invention a standardportion size may be taken as about 250 ml or about 250 g as appropriate.

For the purposes of the invention the GI of a foodstuff is defined asthe iAUC of a blood glucose response curve over about 120 mins afterconsumption of a 50 g available-carbohydrate portion of a foodstuffexpressed as a percentage of that after 50 g oral glucose. For thepurposes of the invention available carbohydrate is that fraction ofcarbohydrate that is absorbed across the gastrointestinal tract andenters into intermediary metabolism. It does not include dietary fibre.

Expressed alternatively still, “sugar depleted” means that a juiceproduct has a reduced amount of free glucose and optionally a reducedamount of sucrose that together result in the juice product having areduced glycaemic index relative to that of a juice product that hasbeen prepared from the same fruit or vegetable in the same way but whichis not sugar-depleted. The sugar-depleted juice preferably has aglycaemic index which is no more than 75%, e.g. no more than about 70,65, 60, 55, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22,20, 18, 16, 14, 12, 10, 5 or 1%, of that of a juice product that hasbeen prepared from the same fruit or vegetable in the same way but whichis not sugar-depleted.

This may also be expressed as a juice product that has a lower massconcentration ratio of free glucose to non-saccharide soluble componentsand optionally a lower mass concentration ratio of sucrose tonon-saccharide soluble components than the corresponding ratios of ajuice product that has been prepared from the same fruit or vegetable inthe same way but which is not sugar-depleted. In these embodiments“lower” means a mass concentration ratio of free glucose/sucrose(individually and as appropriate) to non-saccharide soluble componentswhich is no more than 75%, e.g. no more than about 70, 65, 60, 55, 50,48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14,12, 10, 5 or 1% of that of a juice product that has been prepared fromthe same fruit or vegetable in the same way but which is notsugar-depleted.

Mass concentration is an indication of the mass of a first substancepresent in a defined mass or volume of a second substance. Massconcentration may therefore be expressed as grams per litre (“g/l”),grams per kilogram (“g/kg”), parts-per-million (ppm, i.e. mg of soluteper litre of solvent); “% w/v”; “% w/w, “g/100 ml”; or the like.

More specifically, “sugar-depleted” means that the mass concentrationsof free glucose and optionally sucrose are no more than about 75% of themass concentrations of free glucose and optionally sucrose typicallypresent in the same juice product that has been prepared from the samefruit or vegetable in the same way but which is not sugar-depleted, e.g.no more than about 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1% of the mass concentrations of free glucose and optionally sucrosetypically present in the same juice product that has been prepared fromthe same fruit or vegetable in the same way but which is notsugar-depleted. The extent by which the mass concentrations of freeglucose and optionally sucrose may be lower in the sugar-depleted juiceproduct as compared to a juice product which is not sugar-depleted neednot be the same. Therefore, the mass concentration of free glucose inthe sugar-depleted juice product may be no more than about any one of75, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% the massconcentrations of free glucose typically present in the same juiceproduct that has been prepared from the same fruit or vegetable in thesame way but which is not sugar-depleted, and/or the mass concentrationof sucrose in the sugar-depleted juice product may be no more than aboutany one of 75, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%the mass concentration of sucrose typically present in the same juiceproduct that has been prepared from the same fruit or vegetable in thesame way but which is not sugar-depleted.

More simply “sugar depleted” means the mass concentrations of freeglucose and optionally sucrose in the sugar-depleted juice product is(are independently) reduced by at least about 25, 50, 60, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% as compared to the massconcentrations of free glucose and optionally sucrose in the same juiceproduct that has been prepared from the same fruit or vegetable in thesame way but which is not sugar-depleted.

In the case of a sugar-depleted juice, the above features should beinterpreted by reference to the liquid part of the same fruit orvegetable that has been extracted in a method comprising at least onemechanical means of juice extraction, as defined above.

In certain embodiments the combined mass concentrations of the freeglucose and sucrose in the sugar-depleted juice product of the inventionis no more than about 20 g/l, e.g. no more than about 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01g/l, when said juice product is adjusted in volume with water to give agluconic acid concentration of about 5 g/l or the specificconcentrations disclosed below (e.g. about 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 g/l).

The ratio of free glucose to sucrose within these combined massconcentrations is not restricted and so may be 1:99 to 99:1, e.g. 1:95to 95:1, 1:90 to 90:1, 1:85 to 85:1, 1:80 to 80:1, 1:75 to 75:1, 1:70 to70:1, 1:65 to 65:1, 1:60 to 60:1, 1:55 to 55:1, 1:50 to 50:1, 1:45 to45:1, 1:40 to 40:1, 1:35 to 35:1, 1:30 to 30:1, 1:25 to 25:1, 1:20 to20:1, 1:15 to 15:1, 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1,1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

The above values of combined mass concentration may be considered toapply in the context of a “single strength serving” of thesugar-depleted juice product, i.e. a “ready to serve” or“drinkable/edible” product and in particular a sugar-depleted “not fromconcentrate” juice.

In other embodiments, “sugar-depleted” means that the juice product hasbeen rendered substantially, e.g. essentially, devoid of free glucoseand optionally sucrose.

By “substantially devoid of free glucose” it is meant that thesugar-depleted juice product of the invention contains no more thanabout 5 g/l free glucose, e.g. no more than about 4, 3, 2, 1, 0.5, 0.1,0.05, 0.01 g/l free glucose, when said juice product is adjusted involume with water to give a gluconic acid concentration of about 5 g/lor the specific concentrations disclosed below.

By “substantially devoid of sucrose” it is meant that the sugar-depletedjuice product of the invention contains no more than about 5 g/lsucrose, e.g. no more than about 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 g/lsucrose, when said juice product is adjusted in volume with water togive a gluconic acid concentration of about 5 g/l or the specificconcentrations disclosed below.

By “essentially devoid of free glucose” it is meant that thesugar-depleted juice product of the invention contains a trace amount offree glucose. This may also be expressed as essentially undetectablewith standard analytical means, or at the limit of detection with suchmeans. These measures preferably take place when said juice product isadjusted in volume with water to give a gluconic acid concentration ofabout 5 g/l or the specific concentrations disclosed below. Detectionmay be by any convenient means, e.g. the Reflectoquant™ system of MerckMillipore™ as disclosed in the Examples.

“Essentially devoid of sucrose” should be interpreted in the same way.

The above values for free glucose and sucrose may be considered to applyin the context of a “single strength serving” of the sugar-depletedjuice product, i.e. a “ready to serve” or “drinkable/edible” product andin particular a sugar-depleted “not from concentrate” juice.

The terms “free glucose-depleted” and “sucrose-depleted” should beinterpreted consistently with the foregoing.

In certain embodiments a sugar-depleted juice product of the inventionmay have an increased mass concentration ratio of other sugars, e.g.fructose, to non-saccharide soluble components than the correspondingratios of a juice product that has been prepared from the same fruit orvegetable in the same way but which is not sugar-depleted.

In other embodiments the sugar-depleted juice product may have also beenrendered devoid, or at least have a reduced content or be depleted, ofother sugars, e.g. fructose, typically present in the same juice productthat has been prepared from the same fruit or vegetable in the same waybut which is not sugar-depleted. The above embodiments relating to freeglucose and/or sucrose can be applied mutatis mutandis in the context ofthe depletion of other sugars, e.g. fructose.

Therefore, in a further embodiment of the present invention there isprovided a sugar-depleted fruit or vegetable juice product, wherein saidjuice product is a fruit or vegetable juice or juice-retaining fruit orvegetable derived matter, and wherein said juice product issubstantially, e.g. essentially, devoid of free glucose and sucrose,said juice product contains at least about 5 g/l gluconic acid and saidjuice product contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1 g/l K⁺, and    -   (iii) at least about 0.1 g/l Mg²⁺.

In preferred embodiments the sugar-depleted juice product contains atleast 6 g/l, e.g. at least about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75 or 80 g/lgluconic acid.

In preferred embodiments the sugar-depleted juice product may contain atleast about 0.5 g/l Ca²⁺, e.g. at least about 0.6, 0.7, 0.8, 0.9, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 g/l Ca²⁺.

In preferred embodiments the sugar-depleted juice product may contain atleast about 1 g/l K⁺, e.g. at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5 or 15 g/l K⁺.

In preferred embodiments the sugar-depleted juice product may contain atleast about 0.1 g/l Mg²⁺, e.g. at least about 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 g/l Mg²⁺.

All ratios of Ca²⁺, K⁺ and/or Mg²⁺ mass concentrations which may beformed from the above values are expressly contemplated.

In certain embodiments the sugar-depleted juice product contains Ca²⁺and K⁺ at the above described concentrations. In other embodiments theselected metal ions are Ca²⁺ and Mg²⁺, Mg²⁺ and K⁺, or Ca²⁺, K⁺ and Mg²⁺all at the above described concentrations.

In certain embodiments the juice product of the invention comprises anytwo or three of Ca²⁺, K⁺ and Mg²⁺ at mass concentrations which, whensaid juice product is adjusted in volume with water to give a gluconicacid concentration of about 5 g/l, or the specific concentrationsdisclosed above, gives the above recited values.

The concentration of metal ions referred to herein are concentrations asmay be determined by the physiochemical pressure digestion methoddescribed in EN 13805 (2013). Should alternative approaches be used tomeasure the concentration of metal ions in the juice product, an acid(e.g. nitric acid) digestion step, equivalent to that of EN 13805, mustbe incorporated immediately prior to analysis.

In further preferred embodiments the sugar-depleted juice product is apalatable (“drinkable” or “edible”, “ready to serve”, “single strength”)juice product, e.g. a juice replacement beverage, that has the same orsubstantially the same taste and flavour profile and mouth-feel as anunmodified juice product from the same fruit or vegetable. Such aproduct may be obtained by diluting with water a sugar-depleted juiceproduct of the invention that is more concentrated (contains less water)than the palatable form. It is also preferred that the sugar-depletedjuice product is a sugar-depleted “not from concentrate” juice.

A suitably dilute juice that is palatable to the average consumer maybea juice that corresponds to that obtained directly from the fruit orvegetable in question by a method comprising at least one mechanicalmeans of juice extraction as defined herein. Conveniently this may beexpressed as a sugar-depleted juice having a Brix level (soluble solidscontent) within 30%, e.g. within 25%, 20%, 15%, 10% or 5% of that ofjuice obtained directly from the fruit or vegetable in question by amethod comprising at least one mechanical means of juice extraction asdefined herein. The skilled person would be aware that in the context ofthe present invention the Brix value for a sugar-depleted juice of theinvention is a value representing the sum of sugars including gluconicacid.

The CODEX General Standard For Fruit Juices And Nectars (CODEX STAN247-2005) is incorporated by reference in this regard as a resource forBrix (soluble solids content) thresholds for palatable concentrations ofcommon juices and methodology to calculate the same.

In these embodiments gluconic acid will typically be present in thesugar-depleted juice product at a mass concentration of about 5 to about100 g/l, e.g. about any one of 10, 15, 20, 25, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 60, 65, 70, 75, 80, 85, 90, or 95 toabout 100 g/l, preferably about any one of 15, 20, 25, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65, 70, 75, 80 or 85 toabout 90 g/l, more preferably about any one of 20, 25, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 65 or 70 to about 80 g/l,and still more about preferably about any one of 25, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 to about 75 g/l.

Any range which may be formed from any of the above recited massconcentrations is expressly contemplated.

In other embodiments gluconic acid will typically be present in thesugar-depleted juice product at a mass concentration of about 10 toabout 70 g/l, e.g. about any one of 15, 20, 25, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 60, 65 g/l to about 70 g/l, preferablyabout any one of 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56 or 60 to about 65 g/l, more preferably about any one of 25, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56 to about 60 g/l,and still more preferably about any one of 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54 or 56 to about 60 g/l.

Any range which may be formed from any of the above recited massconcentrations is expressly contemplated.

Gluconic acid content may be measured by any convenient means, e.g. viaan appropriate enzymatic assay (e.g. as supplied by R-Biopharm™) orchromatography based techniques. Such measurements should take intoaccount all forms of gluconic acid, i.e. the free acid, salts thereofand the lactone forms thereof, e.g. D-gluconic acid δ-lactone andD-gluconic acid γ-lactone.

As detailed later, because the preferred method of producing thesugar-depleted juice product of the invention is based on the conversionof at least a portion of the free glucose and optionally the glucose inthe sucrose present in a natural juice product to gluconic acid, theexact amount of gluconic acid present in these embodiments will be to anextent dictated by the amounts of free glucose and glucose in sucroseconverted to gluconic acid. Ultimately the amount of gluconic acidpresent in these embodiments will be dictated by the total free glucoseand sucrose content of the corresponding natural juice product. As alsodetailed later, in other embodiments the free fructose and, optionally,fructose in the sucrose in a natural fruit juice product may beconverted to glucose and thereby contribute to the amount of gluconicacid in the sugar-depleted juice.

In some instances it might be necessary to add gluconic acid to thesugar-depleted juice product to reach the concentrations required by theinvention if the amount of free glucose and the glucose in sucroseavailable is not sufficiently high or if the intended reduction inglucose and sucrose levels is not sufficiently great.

In these embodiments the sugar-depleted juice product may contain about0.5 to about 10 g/l Ca²⁺, e.g. about any one of 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5 or 5 to about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or9.5 g/l Ca²⁺, preferably about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5,or 4 to about any one of 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 g/l Ca²⁺, morepreferably about any one of 0.5, 1, 1.5, 2, 2.5 or 3, to about any oneof 3.5, 4, 4.5, 5, 5.5 or 6 g/l Ca²⁺, and still more preferably aboutany one of 0.5, 1, 1.5, 2 or 2.5 to about any one of 3, 3.5, 4, 4.5 or 5g/l Ca²⁺. Any range which may be formed from any of the above recitedmass concentrations is expressly contemplated.

In these embodiments the sugar-depleted juice may contain about 1 toabout 20 g/l K⁺, e.g. about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10to about any one of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 g/l K⁺,preferably about any one of 1, 2, 3, 4, 5, 6, 7 or 8 to about any one of9, 10, 11, 12, 13, 14, 15 or 16 g/l K⁺, more preferably about any one of1, 2, 3, 4, 5, or 6 to about any one of 7, 8, 9, 10 11 or 12 g/l K⁺, andstill more preferably about any one of 1, 2, 3, 4, or 5 to about any oneof 6, 7, 8, 9 or 10 g/l K⁺. Any range which may be formed from any ofthe above recited mass concentrations is expressly contemplated.

In these embodiments the sugar-depleted juice may contain about 0.1 toabout 2 g/l Mg²⁺, e.g. about any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9 or 1 to about any one of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9 or 2 g/l Mg²⁺, preferably about any one of 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7 or 0.8 to about any one of 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5 or 1.6 g/l Mg²⁺, more preferably about any one of 0.1, 0.2,0.3, 0.4, 0.5 or 0.6 to about any one of 0.7, 0.8, 0.9, 1 1.1 or 1.2 g/lMg²⁺, and still more preferably about any one of 0.1, 0.2, 0.3, 0.4 or0.5, to about any one of 0.6, 0.7, 0.8, 0.9 or 1 g/l Mg²⁺. Any rangewhich may be formed from any of the above recited mass concentrations isexpressly contemplated.

All ratios of Ca²⁺, K⁺ and/or Mg²⁺ mass concentrations which may beformed from the above values are expressly contemplated.

In these embodiments the sugar-depleted juice product has a pH of equalto or greater than about 3 and equal to or less than about 5, e.g. about3 to about 5, about 3.5 to about 4.8, about 3.6 to about 4.6, about 3.8to about 4.4, about 3.8 to about 4.6, about 3.9 to about 4.5 or about4.0 to about 4.5.

Thus, the present invention provides a sugar-depleted fruit or vegetablejuice product, wherein said juice product is a fruit or vegetable juiceor juice-retaining fruit or vegetable derived matter, preferably whereinsaid juice product is substantially, e.g. essentially, devoid of glucoseand sucrose, wherein said juice product contains at least about 5 g/lgluconic acid and wherein said juice product contains any two or three,of Ca²⁺, K⁺, and Mg²⁺ at a mass concentration which, when said juiceproduct is adjusted in volume with water to give a gluconic acidconcentration of about 5 g/l or the specific concentrations describedabove, gives:

-   -   (i) a mass concentration of Ca²⁺ of about 0.5 to about 10 g/l    -   (ii) a mass concentration of K⁺ of about 1 to about 20 g/l,    -   (iii) a mass concentration of Mg²⁺ of about 0.1 to about 2 g/l.

The above embodiments may be considered to apply in the context of a“single strength serving” of the sugar-depleted juice product, i.e. a“ready to serve” or “drinkable/edible” product. Preferably the juiceproduct is a sugar-depleted fruit or vegetable juice, in particular asugar-depleted “not from concentrate” juice.

The above disclosed ranges of Ca²⁺, K⁺, and Mg²⁺ mass concentrations andpH apply mutatis mutandis to this aspect of the invention.

In other embodiments the above recited values and ranges for Ca²⁺, K⁺,and Mg²⁺ mass concentrations are the amounts by which Ca²⁺, K⁺, and Mg²⁺mass concentrations are increased relative to those present naturally ina juice product which has been prepared from the same fruit or vegetablein the same way. In other words the above recited values and ranges forCa²⁺, K⁺, and Mg²⁺ mass concentrations reflect the amounts of Ca²⁺, K⁺,and Mg²⁺ which have been added or introduced to the juice product or bywhich the juice product has been supplemented.

Thus in certain embodiments the invention provides provided asugar-depleted fruit or vegetable juice product, wherein said juiceproduct is a fruit or vegetable juice or juice-retaining fruit orvegetable derived matter, wherein said juice product contains at leastabout 5 g/l gluconic acid and wherein the mass concentrations of any twoor three of Ca²⁺, K⁺, and Mg²⁺ are increased by

-   -   (i) at least about 0.5 g/l for Ca²⁺,    -   (ii) at least about 1 g/l for K⁺, and    -   (iii) at least about 0.1 g/l for Mg²⁺,        as compared to the mass concentrations of Ca²⁺, K⁺, and Mg²⁺        respectively present naturally in a juice product which has been        prepared from the same fruit or vegetable in the same way.

Any specific embodiments of individual elements of the above embodimentapply mutatis mutandis to this aspect of the invention.

In other embodiments the juice product of the invention may have a Ca²⁺mass concentration which is relative to the mass concentration of Ca²⁺present naturally in a juice product which has been prepared from thesame fruit or vegetable in the same way or in the fruit or vegetablefrom which the juice product is obtained. In these embodiments a naturalCa²⁺ mass concentration of 0 g/l to less than 0.5 g/l in a juice productwhich has been prepared from the same fruit or vegetable in the same wayor in the fruit or vegetable from which the juice product is obtainedwill result in the juice product of the invention having at least about0.5 g/l Ca²⁺. A natural Ca²⁺ mass concentration of 0.5 g/l to less than1 g/l will result in the juice product of the invention having at leastabout 1 g/l Ca²⁺. A natural Ca²⁺ mass concentration of 1 g/l to lessthan 1.5 g/l will result in the juice product of the invention having atleast about 1.5 g/l Ca²⁺. A natural Ca²⁺ mass concentration of 1.5 g/lto less than 2 g/l will result in the juice product of the inventionhaving at least about 2 g/l Ca²⁺. A natural Ca²⁺ mass concentration of 2g/l to less than 2.5 g/l will result in the juice product of theinvention having at least about 2.5 g/l Ca²⁺. A natural Ca²⁺ massconcentration of 2.5 g/l to less than 3 g/l will result in the juiceproduct of the invention having at least about 3 g/l Ca²⁺. A naturalCa²⁺ mass concentration of 3 g/l to less than 3.5 g/l will result in thejuice product of the invention having at least about 3.5 g/l Ca²⁺. Anatural Ca²⁺ mass concentration of 3.5 g/l to less than 4 g/l willresult in the juice product of the invention having at least about 4 g/lCa²⁺.

In other embodiments the juice product of the invention may have a K⁺mass concentration which is relative to the mass concentration of K⁺present naturally in a juice product which has been prepared from thesame fruit or vegetable in the same way or in the fruit or vegetablefrom which the juice product is obtained. In these embodiments a naturalK⁺ mass concentration of 0 g/l to less than 1 g/l in a juice productwhich has been prepared from the same fruit or vegetable in the same wayor in the fruit or vegetable from which the juice product is obtainedwill result in the juice product of the invention having at least about1 g/l K⁺. A natural K⁺ mass concentration of 1 g/l to less than 1.5 g/lwill result in the juice product of the invention having at least about1.5 g/l K⁺. A natural K⁺ mass concentration of 1.5 g/l to less than 2g/l will result in the juice product of the invention having at leastabout 2 g/l K⁺. A natural K⁺ mass concentration of 2 g/l to less than2.5 g/l will result in the juice product of the invention having atleast about 2.5 g/l K⁺. A natural K⁺ mass concentration of 2.5 g/l toless than 3 g/l will result in the juice product of the invention havingat least about 3 g/l K⁺. A natural K⁺ mass concentration of 3 g/l toless than 3.5 g/l will result in the juice product of the inventionhaving at least about 3.5 g/l K⁺. A natural K⁺ mass concentration of 3.5g/l to less than 4 g/l will result in the juice product of the inventionhaving at least about 4 g/l K⁺. A natural K⁺ mass concentration of 4 g/lto less than 4.5 g/l will result in the juice product of the inventionhaving at least about 4.5 g/l K⁺. A natural K⁺ mass concentration of 4.5g/l to less than 5 g/l will result in the juice product of the inventionhaving at least about 5 g/l K⁺.

In other embodiments the juice product of the invention may have a Mg²⁺mass concentration which is relative to the mass concentration of Mg²⁺present naturally in a juice product which has been prepared from thesame fruit or vegetable in the same way or in the fruit or vegetablefrom which the juice product is obtained. In these embodiments a naturalMg⁺ mass concentration of 0 g/l to less than 0.1 g/l in a juice productwhich has been prepared from the same fruit or vegetable in the same wayor in the fruit or vegetable from which the juice product is obtainedwill result in the juice product of the invention having at least about0.1 g/l Mg²⁺. A natural Mg²⁺ mass concentration of 0.1 g/l to less than0.15 g/l will result in the juice product of the invention having atleast about 0.15 g/l Mg²⁺. A natural Mg²⁺ mass concentration of 0.15 g/lto less than 0.2 g/l will result in the juice product of the inventionhaving at least about 0.2 g/l Mg²⁺. A natural Mg⁺ mass concentration of0.2 g/l to less than 0.25 g/l will result in the juice product of theinvention having at least about 0.25 g/l Mg²⁺. A natural Mg²⁺ massconcentration of 0.25 g/l to less than 0.3 g/l will result in the juiceproduct of the invention having at least about 0.3 g/l Mg²⁺. A naturalMg²⁺ mass concentration of 0.3 g/l to less than 0.35 g/l will result inthe juice product of the invention having at least about 0.35 g/l Mg²⁺.A natural Mg²⁺ mass concentration of 0.35 g/l to less than 0.4 g/l willresult in the juice product of the invention having at least about 0.4g/l Mg²⁺. A natural Mg²⁺ mass concentration of 0.4 g/l to less than 0.45g/l will result in the juice product of the invention having at leastabout 0.45 g/l Mg²⁺. A natural Mg²⁺ mass concentration of 0.45 g/l toless than 0.5 g/l will result in the juice product of the inventionhaving at least about 0.5 g/l Mg²⁺.

The Ca²⁺, K⁺, and Mg²⁺ mass concentrations present naturally in a juiceproduct which has been prepared from the same fruit or vegetable in thesame way, or in the fruit or vegetable from which the juice product isobtained, can be easily determined as described herein. In addition,databases of food composition, e.g. the McCance and Widdowson'sComposition of Foods Integrated Dataset of Public Health England and theUnited States Department of Agriculture Agricultural Research ServiceNational Nutrient Database for Standard Reference, the contents of whichare incorporated in their entirety by reference, may be consulted.

Any specific embodiments of individual elements of the invention applymutatis mutandis to this aspect of the invention.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from orange, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 2.5 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from apple, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1.5 g/l K⁺, and    -   (iii) at least about 0.1 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from grape, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1.5 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from grapefruit, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 2 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from tomato, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 2.5 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from pineapple, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1.5 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from cranberry, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1 g/l K⁺, and    -   (iii) at least about 0.1 g/l Mg²⁺.

In a further embodiment there is provided a sugar-depleted fruit orvegetable juice product, wherein said juice product is a fruit orvegetable juice or juice-retaining fruit or vegetable derived matter andwherein at least 75%, 80%, 85%, 90%, 95% or essentially all of saidjuice or juice-retaining matter is derived from carrot, wherein saidjuice product contains at least about 5 g/l gluconic acid and said juiceproduct contains any two or three, of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 3 g/l K⁺, and    -   (iii) at least about 0.15 g/l Mg²⁺.

Any specific embodiments of individual elements of the inventiondescribed above apply mutatis mutandis to this aspect of the invention,in particular the specific values and ranges for Ca²⁺, K⁺ and Mg²⁺ massconcentrations. In certain embodiments the above specific juice productsof the invention comprises any two or three of Ca²⁺, K⁺ and Mg²⁺ at massconcentrations which, when said juice product is adjusted in volume withwater to give a gluconic acid concentration of about 5 g/l, or thespecific concentrations disclosed above, gives the above recited values.

The precise combination of metal ion concentrations in any particularembodiment will be to an extent dictated by the overall composition ofthe juice product that has been sugar-depleted in accordance with theinvention. For instance, a juice product from a citrus fruit mightrequire a different combination of metal ion concentration to thatrequired by a juice product from carrots. The skilled person would alsoappreciate that adjustment and optimisation may be required to allow fornatural variations between juice product batches on account ofdifferences in the source of the fruit/vegetables, the overall ripenessof the fruit and the varieties of fruit used to prepare the juiceproduct. In the present invention it is assumed that the sensoryproperties of the sugar-depleted juice product are essentiallyequivalent to a juice product obtained from adequately ripefruit/vegetables, i.e. fruit and vegetables that if juiced would giverise to a palatable beverage. The skilled person would be easily able todetermine workable and optimal combinations of metal ion concentrationsfor his particular juice product of interest in accordance with theinvention, i.e. those that give rise to a sugar-depleted juice productthat is highly palatable and which retains sufficiently the flavour andmouth-feel of the unmodified juice product. Routine techniques such asthe use of trained sensory assessors can be employed in this regard.

The sugar-depleted juice products of the present invention may beprepared through one or more enzyme treatments and metal ionsupplementation. Specifically, a treatment with an enzyme that convertsglucose into gluconic acid (e.g. glucose oxidase), an optional treatmentwith an enzyme which hydrolyses sucrose to glucose and fructose (e.g.invertase) and supplementation with one or more, preferably any two orthree, of a source of Ca²⁺, a source of Mg²⁺ and a source of K⁺ inamounts sufficient to reach the amounts Ca²⁺, Mg²⁺ and K⁺ required bythe sugar-depleted juice product of the invention. Preferably thesugar-depleted juice products of the present invention are not preparedby a method comprising a step in which the juice product is contactedwith a glucose-fructose oxidoreductase, e.g. the glucose-fructoseoxidoreductase from Zymomonas mobilis. In such embodiments thesugar-depleted juice products of the present invention comprise sorbitolin the same amounts or less than those in the same juice product thathas been prepared from the same fruit or vegetable in the same way butwhich is not sugar-depleted.

In other embodiments the sugar-depleted juice product of the inventionis also substantially, e.g. essentially, devoid of fructose, which termis to be interpreted as discussed above for glucose and sucrose. Thismay be conveniently achieved by further treating the sugar-depletedjuice product of the invention with an enzyme capable of convertingfructose into a derivative form, preferably a derivative form with alower calorific value and/or more favourable glycaemic profile. Suchenzymes may include 5-D-fructose dehydrogenase (e.g. as described in US2009/0214620). Alternatively, fructose may be enzymatically converted toglucose prior to or concurrent with treatment with glucose oxidase. Suchenzymes may be defined as glucose isomerases and includeglucose-6-phosphate isomerase and D-xylose isomerase (e.g. as describedin US 2009/0311232). In these embodiments in may be necessary tosupplement the sugar-depleted juice product with a sweetening agent,e.g. an artificial sweetening agent that is not a sugar (e.g. stevia,sucralose and aspartame).

In certain embodiments the sugar-depleted juice product of the inventiondoes not contain detectable amounts of active glucose oxidase, and/orinvertase and/or 5-D-fructose dehydrogenase and/or a glucose isomerase(e.g. glucose-6-phosphate isomerase or D-xylose isomerase). Expressednumerically the sugar-depleted juice product of the invention displaysenzyme activities for the above enzymes of no more than 1 U/ml, e.g. nomore than 0.1 U/ml, 0.05 U/ml or 0.01 U/ml. Nevertheless, the juiceproduct of the invention may still comprise inactivated, e.g. denatured,forms of one or more of the above-mentioned enzymes, e.g. glucoseoxidase and/or invertase. In certain embodiments the sugar-depletedjuice product of the invention does not contain detectable amounts of aglucose-fructose oxidoreductase, e.g. the glucose-fructoseoxidoreductase from Zymomonas mobilis, whether active, inactive,denatured or otherwise.

The sugar-depleted juice product of the invention may be supplementedwith other compounds to enhance the palatability of the product, e.g. byenhancing taste, flavour and mouth-feel. Such compounds include, but arenot limited to other metal ions (e.g. Na⁺, Fe²⁺, Fe³⁺), vitamins (e.g.vitamins A, B, C, D, E, K and subtypes thereof), minerals (e.g.compounds containing phosphorous, sulphur, fluorine, chlorine, boron,chromium, cobalt, copper, iron, manganese, molybdenum, selenium,silicon, tin, vanadium and zinc), flavourings (natural and artificial),flavour enhancers (e.g. monosodium glutamate), preservatives, artificialsweeteners (e.g. stevia, sucralose and aspartame), polyphenols, organicacids (other than gluconic acid), acidity regulators and stabilisers.However, in other embodiments the sugar-depleted juice product of theinvention does not contain the above classes of additives in quantitiesgreater than those found naturally in the unmodified juice product.

In preferred embodiments the sugar-depleted juice product of theinvention contains only the sodium present naturally in the unmodifiedjuice product. Expressed numerically, in preferred embodiments thesugar-depleted juice product of the invention contains Na⁺ at a massconcentration which, when said juice product is adjusted in volume withwater to give a gluconic acid concentration of about 5 g/l, or thespecific concentrations disclosed above, gives a mass concentration ofNa⁺ of no more than about 0.5 g/l, e.g. no more than about 0.45, 0.40,0.35, 0.30, 0.25 or 0.20 g/l.

The term “fruit” is used herein in its culinary sense; that is toencompass any sweet, edible part of a plant, even if it does not developfrom a floral ovary, that has a liquid part that may be extracted bymechanical means and which is safely edible by a human. For the purposesof the invention, this does not include nuts and grains. For thepurposes of the invention this does include tomato. Industriallyrelevant examples include but are not limited to the pome fruits (e.g.apple, pear, rosehip, medlar, quince); the citrus fruits (e.g. orange,blood orange, grapefruit, tangerine, clementine, mandarin, lemon, lime,kumquat, pomelo); the Rubus fruits (e.g. raspberry, blackberry,dewberry, boysenberry, olallieberry, tayberry, cloudberry, loganberry,salmonberry, thimbleberry, wineberry); the Ribes fruits (e.g.blackcurrant, redcurrant, white currant, gooseberry); the Ericaceaefruits (e.g. blueberry, bilberry, cranberry, bearberry, crowberry,falberry, huckleberry, lingonberry); the Prunus stone fruits (e.g.peach, plum, nectarine, apricot, greengage, cherry, damson); grapes,pomegranate, fig, passion fruit, guava, mango, melon, pineapple,elderberry, rhubarb, soursop, tamarind, strawberry, kiwifruit, lychee,papaya, banana, advocado and tomato.

The term “vegetable” is used herein in its culinary sense; that is toencompass any savoury, edible part of a plant, even if it does developfrom a floral ovary, that has a liquid part that may be extracted bymechanical means and which is safely edible by a human. Industriallyrelevant examples include but are not limited to leaf vegetables (e.g.spinach, wheatgrass, choi, lettuce, cabbage, kale, cress, chard); rootvegetables (e.g. carrot, parsnip, potato, yam, beetroot, burdock,ginger, galangal, Jerusalem artichoke, turnip, radish); bulb and stemvegetables (e.g. onion, garlic, asparagus, leek, fennel, celery,lemongrass); and podded vegetables (pea, runner bean, green bean, broadbean). Carrot, wheatgrass, celery, beetroot, ginger and spinach are ofnote.

The sugar-depleted juice product may be a mixture of juice products fromdifferent fruits and/or vegetables. Said juice products may be blendedonce each has been rendered sugar-depleted or may be blended in theirnatural sugar-complete state and subsequently rendered sugar-depleted.

Preferably the juice product is a fruit juice product or a mixture offruit juice products selected from apple, pear, orange, grapefruit,mandarin, tangerine, clementine, lemon, lime, grape, pineapple, mango,guava, soursop, tomato, pomegranate, cranberry, blueberry, blackcurrant,passion fruit, rhubarb, melon, strawberry, raspberry, peach, nectarine,apricot and cherry.

As mentioned above, the sugar-depleted juice product of the presentinvention may be prepared through one or more enzyme treatments andmetal ion supplementation. Specifically, an enzyme treatment thatconverts glucose into gluconic acid (e.g. glucose oxidase), an optionaltreatment with an enzyme which hydrolyses sucrose to glucose andfructose (e.g. invertase) and supplementation with one or more,preferably any two or three, of a source of Ca²⁺, a source of Mg²⁺ and asource of K⁺ in amounts sufficient to reach the amounts Ca²⁺, Mg²⁺ andK⁺ required by the sugar-depleted juice product of the invention. Thejuice product undergoing the treatments of the invention may be inconcentrated form or diluted form. In other embodiments thesugar-depleted juice product that has been prepared in accordance withthe invention may undergo concentration or dilution. In still furtherembodiments, concentration and/or dilution steps may interspace thetreatment steps of the invention.

Thus, in another aspect of the invention, there is provided a method forthe preparation of a sugar-depleted fruit or vegetable juice product,wherein said sugar-depleted juice product is a fruit or vegetable juiceor juice-retaining fruit or vegetable derived matter, wherein saidsugar-depleted juice product contains at least about 5 g/l gluconic acidand wherein said sugar-depleted juice product contains any two or three,of

-   -   (i) at least about 0.5 g/l Ca²⁺,    -   (ii) at least about 1 g/l K⁺, and    -   (iii) at least about 0.1 g/l Mg²⁺,        i.e. any of the sugar-depleted juice products disclosed herein,        said method comprising providing a fruit or vegetable juice        product containing free glucose and/or sucrose and:    -   (a) contacting said juice product with an enzyme which        hydrolyses sucrose to glucose and fructose,    -   (b) contacting the enzyme treated juice product of step (a) with        an enzyme which converts glucose into gluconic acid, and    -   (c) supplementing said juice product with one or more,        preferably any two or three, of a source of Ca²⁺, a source of        Mg²⁺ and a source of K⁺ in an amount sufficient to give said        mass concentrations of Ca²⁺, Mg²⁺ and/or K⁺, respectively,        wherein steps (a) and/or (b) may be performed simultaneously        with step (c) or before or after step (c).

In other embodiments said method comprises providing a sucrose-depletedfruit or vegetable juice product containing free glucose and:

-   -   (d) contacting said juice product with an enzyme which converts        glucose into gluconic acid, and    -   (e) supplementing said juice product with one or more,        preferably any two or three, of a source of Ca²⁺, a source of        Mg²⁺ and a source of K⁺ in an amount sufficient to give said        mass concentrations of Ca²⁺, Mg²⁺ and/or K⁺, respectively,        wherein steps (d) and (e) may be performed simultaneously or        separately in any order.

In other embodiments said method comprises providing a sucrose-depletedfruit or vegetable juice product containing free glucose and said massconcentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺, respectivelyand:

-   -   (f) contacting said juice with an enzyme which converts glucose        into gluconic acid.

In other embodiments said method comprises providing a freeglucose-depleted fruit or vegetable juice product, wherein said juiceproduct contains at least about 5 g/l gluconic acid and:

-   -   (g) contacting said juice product with an enzyme which        hydrolyses sucrose to glucose and fructose,    -   (h) supplementing said juice product with one or more,        preferably any two or three, of a source of Ca²⁺, a source of        Mg²⁺ and a source of K⁺ in an amount sufficient to give said        mass concentrations of Ca²⁺, Mg²⁺ and/or K⁺, respectively,        wherein steps (g) and (h) may be performed simultaneously or        separately in any order.

In other embodiments said method comprises providing a freeglucose-depleted fruit or vegetable juice product, wherein said juiceproduct contains at least about 5 g/l gluconic acid and said massconcentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺, respectivelyand:

-   -   (i) contacting said juice product with an enzyme which        hydrolyses sucrose to glucose and fructose.

In other embodiments said method comprises providing a free glucose andoptionally sucrose depleted fruit or vegetable juice product, whereinsaid juice product contains at least about 5 g/l gluconic acid and:

-   -   (j) supplementing said juice product with one or more,        preferably any two or three, of a source of Ca²⁺, a source of        Mg²⁺ and a source of K⁺ in an amount sufficient to give said        mass concentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺,        respectively.

In other embodiments said method comprises providing a fruit orvegetable juice product containing free glucose and/or sucrose and saidmass concentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺,respectively, and:

-   -   (k) contacting said juice product with an enzyme which        hydrolyses sucrose to glucose and fructose, and    -   (l) contacting the enzyme treated juice of step (k) with an        enzyme which converts glucose into gluconic acid,        wherein steps (k) and (l) may be performed simultaneously or        separately.

In other embodiments said method comprises providing a fruit orvegetable juice product containing free glucose and optionally sucrose,and:

-   -   (m) contacting said juice product with an enzyme which converts        glucose into gluconic acid, and    -   (n) supplementing said juice product with one or more,        preferably any two or three, of a source of Ca²⁺, a source of        Mg²⁺ and a source of K⁺ in an amount sufficient to give said        mass concentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺,        respectively,        wherein steps (m) and (n) may be performed simultaneously or        separately in any order.

In other embodiments said method comprises providing a fruit orvegetable juice product containing free glucose and optionally sucroseand said mass concentrations of any two or three of Ca²⁺, Mg²⁺ and/orK⁺, respectively, and:

-   -   (o) contacting said juice product with an enzyme which converts        glucose into gluconic acid.

In these embodiments the final step recited therein is sufficient toresult in the formation of a sugar-depleted juice product of theinvention. However, further processing steps may be included before orfollowing or intervening the recited steps and the final product maystill be considered a sugar-depleted juice product of the invention tothe extent such products are defined herein.

In these embodiments the term “said juice product” or “the juiceproduct” should be construed according to immediate context and takinginto account preceding treatment steps in all possible sequences, unlessthe term is directly preceded or directly followed by the qualifyingterm “sugar-depleted”, in which case reference is being made to thesugar-depleted juice products of the invention as described herein.

Preferably in these embodiments “depleted” means substantially, e.g.essentially, devoid as defined herein.

Preferably the enzyme which hydrolyses sucrose to glucose and fructoseis an invertase or a sucrase (e.g. sucrase-isomaltase and sucrosealpha-glucosidase).

Preferably the enzyme which converts glucose to gluconic acid is aglucose oxidase. Preferably the enzyme which converts glucose togluconic acid is not a glucose-fructose oxidoreductase, e.g. theglucose-fructose oxidoreductase from Zymomonas mobilis.

The step of contacting with the enzyme which hydrolyses sucrose toglucose and fructose is performed for a time and with an amount ofenzyme that, under the physical conditions used (e.g. pH, temperature,pressure and oxygen concentration), are sufficient to hydrolyse asufficient amount of sucrose in the sample to glucose and fructose torender the sample sucrose-depleted. Preferably, the step of contactingwith the enzyme which hydrolyses sucrose to glucose and fructose isperformed for a time and with an amount of enzyme that, under thephysical conditions used, are sufficient to hydrolyse substantially,e.g. essentially, all of the sucrose in the sample to glucose andfructose, i.e. to render the sample substantially, e.g. essentially,devoid of sucrose.

In embodiments in which invertase is selected as the enzyme to hydrolysesucrose to glucose and fructose, the enzyme will typically be used at aconcentration of 500-50000 U/l, preferably 1000-10000 U/l, mostpreferably at about 5000 U/l, e.g. 4500-5500 U/l and the invertaseshould be allowed to incubate with the sample for up to 48 hours,preferably 6-48, 6-36, 6-24 or 6-20 hours, most preferably 8-12 hours ata temperature of 5 to 30° C., e.g. 10 to 28° C., 16 to 24° C. or aboutroom temperature (20° C.). Typically, this step of the methods of theinvention will be conducted at atmospheric pressure, e.g. about 70 kPato about 105 kPa. Typically this step of the methods of the inventionwill be conducted at an acidic to neutral pH, e.g. about 2 to about 8,about 3 to about 7, about 4 to about 6, about 4 to about 5, e.g. around4.5. These conditions may also be applied generally in the context ofthe enzyme used to hydrolyse sucrose to glucose and fructose.

The step of contacting with the enzyme which converts glucose togluconic acid is performed for a time and with an amount of enzyme that,under the physical conditions used (e.g. temperature, pressure andoxygen concentration), are sufficient to convert a sufficient amount offree glucose in the sample to gluconic acid to render the sampleglucose-depleted. Preferably, the step of contacting with the enzymewhich converts glucose to gluconic acid is performed for a time and withan amount of enzyme that, under the physical conditions used, aresufficient to convert substantially, e.g. essentially, all of the freeglucose in the sample to gluconic acid, i.e. to render the samplesubstantially, e.g. essentially, devoid of free glucose. Preferably theaction of the enzyme which converts glucose to gluconic acid (e.g.glucose oxidase) results in the requisite amounts of gluconic acid.

In embodiments in which glucose oxidase is selected as the enzyme toconvert glucose to gluconic acid, the enzyme will typically be used at aconcentration 300-30000 U/l, more preferably 1000-10000 U/l, mostpreferably at about 3000 U/l, e.g. 2500-3500 U/l and the glucose oxidaseshould be allowed to incubate with the sample for up to 48 hours,preferably 2-48, 2-36, 2-24, 2-18, 2-12 or 2-10 hours, most preferably3-4 hours at a temperature of 5 to 30° C., e.g. 10 to 28° C., 16 to 24°C. or about room temperature (20° C.). Typically, this step of themethods of the invention will be conducted at atmospheric pressure, e.g.about 70 kPa to about 105 kPa. These conditions may also be appliedgenerally in the context of the enzyme used to convert glucose togluconic acid.

The presence of oxygen is necessary for glucose oxidase to effectivelyconvert glucose into gluconic acid. The treatment of certain juices inaccordance with the invention may therefore benefit from oxygensupplementation in order to ensure, optimal enzyme activity and/orconversion of sufficient amounts of glucose to gluconic acid.Accordingly, in another preferred embodiment of the present invention,oxygenation is performed at least during the glucose oxidase treatmentstep. The oxygen may be supplied in the form of air, but pure oxygen(O₂) is preferable since the process of enzymatic conversion of glucoseto gluconic acid is tends to be faster when pure oxygen is supplied.

The oxygenation of the juice product may result in the oxidation ofascorbic acid (vitamin C). Thus, in these embodiments the fruit juiceproduct of the invention may be substantially, e.g. essentially, devoidof the reduced form of ascorbic acid. By “substantially devoid ofascorbic acid” it is meant that the sugar-depleted juice product of theinvention contains no more than about 10 mg/l ascorbic acid, e.g. nomore than about 8, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 mg/l ascorbicacid, when said juice product is adjusted in volume with water to give agluconic acid concentration of about 5 g/l or the specificconcentrations disclosed above. By “essentially devoid of ascorbic acid”it is meant that the sugar-depleted juice product of the inventioncontains a trace amount of ascorbic acid. This may also be expressed asessentially undetectable with standard analytical means, or at the limitof detection with such means. These measures preferably take place whensaid juice product is adjusted in volume with water to give a gluconicacid concentration of about 5 g/l or the specific concentrationsdisclosed below. Detection may be by any convenient means, e.g. theReflectoquant™ system of Merck Millipore™ as disclosed in the Examples.In other embodiments however the juice product contains ascorbic acid,e.g. at a mass concentration about equal to or above that of theunmodified juice product, i.e. the juice product has been supplementedwith ascorbic acid e.g. following (or in some embodiments during orafter) the oxygenation of the juice product during its production.

A by-product of the conversion of glucose to gluconic acid by glucoseoxidase is hydrogen peroxide. Accordingly, in a preferred embodimentcatalase or other hydrogen peroxide degrading enzyme is present duringany glucose oxidase treatment step or any glucose oxidase treated sampleis treated with catalase or other hydrogen peroxide degrading enzyme.

Glucose oxidase performs optimally at about pH 3-6 and as such the stepof contacting the juice sample with glucose oxidase will preferably beconducted at about pH 3-6, preferably about 3.4-5, more preferably about3.6-4.6 or 3.6-4.8 and most preferably about 3.8-4.4. These conditionsmay also be applied generally in the context of the enzyme used toconvert glucose to gluconic acid. The pH range may be controlled by anyconvenient means, e.g. by the use of appropriate, acids, bases and/orbuffers. It may be convenient to adjust pH prior to treatment of thesample with glucose oxidase and/or during the treatment itself, in whichcase the pH-adjusting agent(s) may be introduced in a plurality ofapplications. pH may be monitored by any convenient means, e.g. pHmeter. In preferred embodiments, pH is controlled, at least in part,with the source of Ca²⁺, Mg²⁺ and/or K⁺, e.g. hydroxides, oxides orsalts of these metals. As shown in the Examples, MgO (e.g. in the formof a slurry, e.g. a slurry wherein the liquid part is an aliquot of thejuice undergoing treatment), Mg(OH₂), KOH and Ca(OH)₂ (e.g. in solidform, for instance as a powder) may be conveniently used.

The sources of Ca²⁺, Mg²⁺ and/or K⁺ are only restricted insofar as thesources must be compatible with food products, they do not affect theactivities of any enzymes used after their introduction to the juice andthey do not have a detrimental effect on the advantageous properties ofthe sugar-depleted juice product of the invention, i.e. its palatabilityand its favourable glycaemic profile. The skilled person would have notrouble in selecting appropriate sources of Ca²⁺, Mg²⁺ and/or K⁺. By wayof example, suitable sources include but are not limited to salts (e.g.halide salts, including fluoride, chloride, bromide, iodide salts;organic salts, including acetate, citrate, glutamate), oxides,hydroxides, peroxides, sulphates, phosphates, nitrites, nitrates,bicarbonates and carbonates. Oxides, peroxides and hydroxides are ofnote as the inventors have found that these compounds, when added to thejuice product, do not go on to form compounds which may affect theflavour and mouth-feel of the juice product. For such reasonsbicarbonate and carbonate salts of Ca²⁺, Mg²⁺ and/or K⁺ should be usedwith some care and preferably will not be used. In certain embodimentscalcium carbonate in particular is not used. A plurality of differentsources of Ca²⁺, Mg²⁺ and/or K⁺ may be used, e.g. MgO, Mg(OH₂), KOH,K₂O₂, CaO and Ca(OH)₂. Conveniently the sources of Ca²⁺, Mg²⁺ and/or K⁺may be selected to optimise the enzyme treatment steps. For instance,oxides and hydroxides may be selected if the enzyme treatments would beimproved by raising the pH of the reaction mixture. Solid (e.g. powder,granule or pellet) forms of the sources of Ca²⁺, Mg²⁺ and/or K⁺ may beadvantageous as perceptible dilution of the juice product is avoided.

The exact total sugar content of the final sugar-depleted juice productobtained from the methods according to the invention will of course varywith the raw material and the process specifications, but the glucoseand optionally the sucrose levels will be reduced, e.g. to trace levels,and the fructose levels may be increased accordingly (if there has beenconversion of sucrose to glucose and fructose), but will preferablystill be within the possible range for natural variation withindifferent species for the raw material used. The overall sugar levelsmay be lower, preferably lowered by 0-70%, usually 10-50% depending onraw material, and most preferably by 25-35%.

In other embodiments the sugar-depleted juice product of the inventionis also depleted of, e.g. rendered essentially devoid of, free fructose.This may be conveniently achieved by incorporating a step in which thejuice product undergoing treatment is exposed to an enzyme capable ofconverting free fructose into a derivative form, preferably a derivativeform with a lower calorific value and/or more favourable glycaemicprofile. Such enzymes may include 5-D-fructose dehydrogenase.

In the methods of the invention treatment with said enzyme shouldpreferably occur after, although not necessarily directly after, thestep in which sucrose is hydrolysed to glucose and fructose, ortreatment with said enzyme is of a sample that is provided depleted of,e.g. devoid of, sucrose and/or glucose.

Alternatively, free fructose may be enzymatically converted to freeglucose, e.g. prior to or concurrent with treatment with glucoseoxidase. Such enzymes may be define as glucose isomerases and includeglucose-6-phosphate isomerase and D-xylose isomerase.

In a further embodiment the starting material for the above describedmethods is a material that is fructose-depleted, e.g. substantially oressentially devoid of fructose.

Glucose, sucrose, gluconic acid and, if required, fructose may bemonitored in the methods of the invention by any of the numerous routineand convenient techniques available to the skilled person. By way ofexample, the free glucose and the sucrose concentration in juice samplesmay be measured using a rapid and simple reflectometric based kit (e.g.Reflectoquant from Merck) and free fructose and gluconic acid may bedetermined via an appropriate enzymatic assay (e.g. as supplied byR-Biopharm).

Any or all of the enzymes used in the methods of the invention may beused in a form immobilised a solid support, preferably a particulatesolid support, e.g. a magnetic particulate solid support. In this wayrecovery of the enzyme(s) is convenient. Preferably, glucose oxidaseand/or catalase are used in a form immobilised on a solid support,preferably a solid support carrying both glucose oxidase and catalaseimmobilised thereon is employed in the methods of the invention.

In certain embodiments the sugar-depleted juice product of the inventiondoes not contain detectable amounts of an active form of one or more theabove-mentioned enzymes. This may be achieved by mechanical removal ofthe enzymes, e.g. by affinity chromatography or by collecting the enzymelinked solid support if such supports are used. Alternatively oradditionally the sugar-depleted juice product of the invention mayundergo heat treatment to inactivate the enzyme. Conveniently this maytake the form of a pasteurisation process. Thus, in certain embodiments,the juice product of the invention may still comprise inactivated formsof one or more of the above-mentioned enzymes, e.g. an enzyme whichconverts glucose into gluconic acid (e.g. glucose oxidase) and/or anenzyme which hydrolyses sucrose to glucose and fructose (e.g.invertase).

The starting materials for the above described methods may be providedin pasteurised form.

In a further aspect of the invention there is provided a sugar-depletedjuice product obtained or obtainable from the methods disclosed herein.

The sugar-depleted juice product of the invention may of course also beused in the preparation of other food products, e.g. purees, sauces andtoppings, jams, jellies, fruit butters and spreads, dessert products(including frozen desserts such as ice cream), juice based candies orgelatins, mixed juice products such as smoothies or yoghurts, diabetic,low carbohydrate and low calorie products, or dietary supplementscontaining juice. The sugar-depleted juice product of the invention mayalso be used to make alcoholic products such as wine with reducedalcohol content (from grape juice or other juices), either directly fromjuice product according to the present invention by standard wine makingtechniques, or by mixing the juice product with alcohol and optionallyother ingredients.

The presence of gluconic acid in combination with reduced amounts ofsucrose and glucose means the modified juice product of the inventionhas a glycaemic response, a glycaemic index, a glycaemic load and aninsulin response which are significantly lower than those of anuntreated juice, i.e. a more favourable glycaemic profile and loweravailable carbohydrate content, respectively, which in turn reduces theamount of dietary energy provided by the product, and thus renders themodified juice product useful as part of a healthy diet in healthysubjects, and also surprisingly effective in treating subjects with orat risk of developing complex metabolic disorders associated with theover-consumption of glucose and/or sucrose and/or inappropriatemetabolism of glucose including metabolic syndrome, diabetes, obesity,dyslipidemia, insulin resistance, hypertension and liver steatosis. Thesuperior palatability of the sugar-depleted juice product of inventionmeans that the product is a viable alternative to natural fruit juiceproducts that will see enthusiastic adoption and prolonged use byconsumers, resulting in the above beneficial effects in consumers andpatients.

The sugar-depleted juice product of the invention preferably results ina reduced insulin response in a subject as measured by the area underthe curve (AUC) of a subject's blood insulin (preferably venous bloodinsulin) levels over time, preferably over about 15 mins, 30 mins, 45mins, 60 mins, 75 mins, 90 mins, 105 mins, 120 mins, 150 mins, 180 mins,210 mins or 240 mins immediately following consumption of thesugar-depleted juice product, relative to a juice product that has beenprepared from the same fruit or vegetable in the same way but which isnot sugar-depleted. Preferably the AUC is calculated as the incrementalAUC (iAUC), i.e. all area below the curve but above the fasting bloodinsulin concentration. Preferably the AUC, e.g. the iAUC, is calculatedover about 120 mins. The insulin response of each product should bedetermined in the same way.

The sugar-depleted juice product preferably results in an area under theblood insulin curve as defined above in response to its consumption thatis no more than 75%, e.g. no more than about 70, 65, 60, 55, 50, 48, 46,44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10,5 or 1% of that of a juice product that has been prepared from the samefruit or vegetable in the same way but which is not sugar-depleted.

The sugar-depleted juice product of the invention preferably results inenhanced postprandial insulin sensitivity, e.g. as measured by ISIaccording to Belifore (ISI=2/[AUC insulin×AUC glucose+1] relative to ajuice product that has been prepared from the same fruit or vegetable inthe same way but which is not sugar-depleted. The sugar-depleted juiceproduct preferably results in postprandial insulin sensitivity that isat least 2 times, e.g. 3, 4, 5, 6, 7, 8, 9 or 10 times greater than thatof a juice product that has been prepared from the same fruit orvegetable in the same way but which is not sugar-depleted.

Thus, in a further aspect the invention provides a method of assistingin maintaining the health and well-being of a subject or for maintainingor promoting health and well-being in a subject, said method comprisingconsuming a sugar-depleted juice product of the invention. The use ofthe juice products of the invention in such methods is contemplated asis the use of the juice products of the invention in the manufacture ofa nutraceutical or food-substitute for use in such methods.

Complex metabolic conditions associated with the over-consumption ofglucose and/or sucrose and/or inappropriate metabolism of glucose, e.g.metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia,insulin resistance, hypertension and liver steatosis are well known,however successful treatment thereof has remained elusive. It has nowsurprisingly been found that simply reducing a subject's dietary intakeof these sugars does not effectively treat these conditions but that amore fruitful approach is to provide these subjects with afood-substitute having a more favourable glycaemic profile and/orinsulin response and/or the ability to increase insulin sensitivity,specifically a sugar-depleted juice product containing gluconic acid inamounts corresponding to amounts of glucose removed from the juiceproduct, i.e. a sugar-depleted juice product of the invention.

Thus, in a further aspect the invention provides a method for thetreatment or prevention of a disease or condition associated with theover-consumption of glucose and/or sucrose and/or inappropriatemetabolism of glucose, said method comprising administering asugar-depleted juice product of the invention to a subject on acalorie-controlled diet.

Expressed differently, the invention provides a sugar-depleted juiceproduct of the invention for use in the treatment or prevention of adisease or condition associated with the over-consumption of glucoseand/or sucrose and/or inappropriate metabolism of glucose in a subjecton a calorie-controlled diet.

Expressed differently, the invention provides for the use of asugar-depleted juice product of the invention in the manufacture of amedicinal product for use in the treatment or prevention of a disease orcondition associated with the over-consumption of glucose and/or sucroseand/or inappropriate metabolism of glucose in a subject on acalorie-controlled diet.

A calorie-controlled diet is a diet which permits a subject to consume adefined number of calories per day, typically this will be acalorie-restricted diet that permits the subject to consume a number ofcalories per day that is fewer than the number the subject consumedbefore adopting the diet. This may be fewer than the number of caloriesrecommended by the skilled practitioner for the average subject or asubject of equivalent body proportions. Preferably the diet will besugar-controlled/sugar-restricted, in particular will be free glucose-and/or sucrose-controlled/restricted, which terms should be interpretedas for calorie-controlled and calorie restricted.

The disease or condition associated with the over-consumption of glucoseand/or sucrose and/or inappropriate metabolism of glucose may beselected from metabolic syndrome, diabetes mellitus type II, obesity,abdominal obesity, dyslipidaemia, insulin resistance, hyperinsulinemia,impaired glucose metabolism, hypertension, liver steatosis,steatohepatitis, hypertriglyceridemia, hypercholesterolemia, low HDLlevels, high LDL levels, pancreatitis, neurodegenerative disease,retinopathy, nephropathy and neuropathy. Obesity, abdominal obesity,dyslipidaemia, insulin resistance, hyperinsulinemia, impaired glucosemetabolism, hypertension, hypertriglyceridemia, hypercholesterolemia,low HDL levels, high LDL levels, neurodegenerative disease, retinopathy,nephropathy and neuropathy are of note.

Acidification of the sugar-depleted juice and the intestinal milieu bygluconic acid and complex formation with minerals such as calcium,magnesium, potassium, selenium, zinc and iron increases their solubilityand bioavailability. This leads to increased absorption and retention ofthese minerals and consequently the contribution of these minerals tothe on-going health and well-being of a subject is maximised. By way ofexample, calcium plays a role in blood coagulation, energy-yieldingmetabolism, muscle function, neurotransmission, digestive enzymefunction, cell division and differentiation, development and maintenanceof bones and teeth; potassium plays a role in muscular and neurologicalfunction and blood pressure; magnesium plays a role in the reduction oftiredness and fatigue, electrolyte balance, energy-yielding metabolism,neurotransmission, muscle contraction, protein synthesis, psychologicalfunction, maintenance of bones and teeth, cell division; selenium playsa role in spermatogenesis, maintenance of hair and nails, immune systemfunction, thyroid function, protection of DNA, proteins and lipids fromoxidative damage; zinc plays a role in DNA synthesis and cell division,carbohydrate and macronutrient metabolism, cognitive function, fertilityand reproduction, maintenance of serum testosterone concentrations,vitamin A metabolism, protein synthesis, maintenance of bones, hair,nails and skin, immune system function, protection of DNA, proteins andlipids from oxidative damage, DNA synthesis and cell division, and ironplays a role in cognitive function, energy-yielding metabolism,formation of red blood cells and haemoglobin, oxygen transport, immunesystem function, reduction of tiredness and fatigue, cell division andcognitive development of children. The specific levels of Ca²⁺, K⁺ andMg²⁺ in the sugar-depleted juice of the invention further enhance theseeffects.

In particular acidification of the sugar-depleted juice and theintestinal milieu by gluconic acid and complex formation with mineralssuch as calcium, magnesium, potassium, selenium, zinc and iron leads tomineralisation of bone and reduction of blood pressure.

Thus, in a further aspect the invention provides a method for increasingthe absorption and retention of dietary minerals or the mineralisationof bone, said method comprising administering a sugar-depleted juiceproduct of the invention to a subject.

Expressed differently, the invention provides a sugar-depleted juiceproduct of the invention for use in increasing the absorption andretention of dietary minerals or the mineralisation of bone in asubject.

Expressed differently, the invention provides for the use of asugar-depleted juice product of the invention in the manufacture of amedicinal product for use in increasing the absorption and retention ofdietary minerals or the mineralisation of bone in a subject.

In turn the invention provides a method for treating perturbations,caused by insufficient absorbance or retention of dietary minerals, inblood coagulation, energy-yielding metabolism, muscle function,neurotransmission, digestive enzyme function, cell division anddifferentiation, development and maintenance of bones and teeth, bloodpressure, the reduction of tiredness and fatigue, electrolyte balance,protein synthesis, psychological function, spermatogenesis, maintenanceof hair and nails, immune system function, thyroid function, protectionof DNA, proteins and lipids from oxidative damage, DNA synthesis,carbohydrate and macronutrient metabolism, cognitive function, fertilityand reproduction, maintenance of serum testosterone concentrations,vitamin A metabolism, formation of red blood cells and haemoglobin,oxygen transport, and cognitive development of children. In particular,increased mineralisation of bone will lead to the effective treatmentand prevention of bone loss disorders including osteoporosis andarthritis.

Osmotic effects by non-absorbed gluconate as well as short chain fattyacids released from gluconate utilizing intestinal microorganismsaccelerate gastrointestinal transit, soften stools and increase faecalvolume. Hence conversion from sugars to gluconate provides foods witheffects against constipation. The juice products of the invention may beused to treat constipation and slow GI transit.

“Treatment” when used in relation to the treatment of a medicalcondition in a subject in accordance with the invention is used broadlyherein to include any therapeutic effect, i.e. any beneficial effect onthe condition or in relation to the condition. Thus, not only includedis eradication or elimination of the condition, or cure of the subject,but also any improvement in the condition. Thus included for example, isan improvement in any symptom or sign of the condition, or in anyclinically accepted indicator of the condition (for example, animprovement in the metabolism of glucose). Treatment thus includes bothcurative and palliative therapy, e.g. of a pre-existing or diagnosedcondition, i.e. a reactionary treatment.

“Prevention” as used herein refers to any prophylactic or preventativeeffect. It thus includes delaying, limiting, reducing or preventing thecondition or the onset of the condition, or one or more symptoms orindications thereof, for example relative to the condition or symptom orindication prior to the prophylactic treatment. Prophylaxis thusexplicitly includes both absolute prevention of occurrence ordevelopment of the condition, or symptom or indication thereof, and anydelay in the onset or development of the condition or symptom orindication, or reduction or limitation on the development or progressionof the condition or symptom or indication.

Preferably the subject is a human, especially a human suffering from orat risk of developing a disease or conditions recited herein, in otherwords a human subject in need of the treatments disclosed herein.

The invention will be further described with reference to the followingnon-limiting Examples in which:

FIG. 1 shows the measurements of pH and glucose, sucrose, fructose andgluconic acid (g/l) taken during the experiment of Example 1. Invertaseis added at 0 hr [101], Glucose oxidase/catalase and 1.25 g/l MgO isadded at 12 hrs [102], 1 g/l MgO was added at 12 hrs 30 min [103], 0.2g/l MgO was added at 13 hrs 50 min [104] and reaction was stopped at 14hrs 15 min (finished product prior to packaging) [105]. Open squares:fructose; open triangles: free glucose; closed circles: gluconic acid;closed triangles: sucrose; crosses: pH.

FIG. 2 shows (A) incremental capillary blood glucose and (B) incrementalvenous plasma insulin levels before and after oral ingestion of 500 mLcontrol apple juice without (control) and a sugar depleted apple juiceproduct of the invention(verum) in 30 men with impaired fasting glucose(IFG). Least square means (LSM)±SEM.

EXAMPLES Example 1 Preparation of a Sugar Depleted Apple Juice Product

An example of a preferred process and sugar-depleted juice productaccording to the present invention is exemplified by the followingpreparation of an apple juice product. Standard large scale apple juiceproduction techniques were first used to produce an apple juice, whichwas then pasteurised and used in a method according to the presentinvention to produce a sugar-depleted juice product according to thepresent invention. Although the preparation of an apple juice product isexemplified herein, this could of course be modified to produce otherjuice products in accordance with the present invention.

Materials and Methods

A pilot scale production run with three batches has been carried out.Results are shown in FIG. 1 and Table 1. The apples used in theproduction were 17.5% Red Gravenstein (1400 kg) and the remainder Aroma(6600 kg). All the apples were bought from Sognefrukt BA,Sognefjordvegen 130, 6863 Leikanger. The juice was pressed from theapples and pasteurized. Then invertase (Maxinvert L4000, DSM) was addedto 2000 l pasteurized apple juice (0.5 g, 5000 U/l) to split thenon-reducing disaccharide sucrose into fructose and glucose over a 12hour/overnight incubation at ca. 20° C. At the same time a commerciallyavailable pectinase was added at 30 g/1000 l, this is the same asstandard apple juice production process. To regulate pH prior to glucoseoxidase/catalase treatment magnesium hydroxide slurry (Mg(OH)₂), madefrom adding magnesium oxide (MgO) to water, was added until the pH ofthe juice was about 3.8-4.4. The enzymes glucose oxidase/catalase (asHyderase L, Amano/Mitsubishi) were added to the juice (0.2 g/l, 3000U/l) followed by oxygen added and maintained at a concentration in thejuice of 3-4 mg/l. pH and oxygen were monitored (data not shown)throughout the glucose oxidase incubation via an automated computercontrolled system. pH was maintained at 3.8-4.4 by batch addition ofMg(OH)₂ when necessary. A typical incubation time of 3-4 hours at roomtemperature is sufficient to reduce almost all glucose to gluconic acid.Glucose was monitored over the incubation period by measuring theglucose concentration in juice samples using a rapid and simplereflectometric based kit (Reflectoquant from Merck). The sucrose contentafter invertase incubation can also be measured in this way. Fructoseand gluconic acid were determined via enzymatic assay (r-biopharm). Oncemost glucose had disappeared all enzyme activity was terminated bypasteurisation. The final product was referred to as a new apple drink,and is a sugar-depleted juice product in accordance with the presentinvention. A wide range of nutritional and chemical parameters of thefinished and packaged products were also determined within 2 months. Areference apple juice (conventional juice) was also analysedsimultaneously as a control, and it comprised a blend of equal parts ofthe same apple juice (1:1:1 by weight) used as the starting juice ineach pilot scale production batch.

Results

FIG. 1 discloses the experimental results, and is a typical example ofpH, concentration of glucose, sucrose, fructose and gluconic acidmonitored during the enzymatic conversion process to make a new applejuice product in accordance with the present invention. The followingmain findings were shown:

The sucrose content was reduced to levels below the limit of detectionby the action of invertase over a period of 12 hours at roomtemperature. The Limit of Detection is 1 g/L.

The glucose content was slightly increased by the action of invertase onsucrose over a period of 12 hours at room temperature and thensignificantly reduced to remove almost all glucose by the action ofglucose oxidase/over a further 2-3 hour period. Thus the glucose contentis decreased significantly from approximately 14 g/L in the conventionalcontrol juice to less than 3 g/L in the packaged juice product (meanvalue of 1.8 g/L over 3 batches tested).

The fructose content was slightly increased by the action of invertaseon sucrose over a period of 12 hours at room temperature, and thenremained constant during the rest of the production process. Thus, thefructose content in the packaged juice increased by approximately 20%(from approximately 58.2 g/L in the conventional juice) to a mean of68.7 g/L over 3 batches tested. The extra fructose in the finished andpackaged apple juice product may be desirable because it contributes tomaintain sweetness.

The sum of sugars in the finished and packaged apple juice product (mean70.6 g/L over 3 batches) is less than that in the conventional applejuice (96.6 g/L), equating to a reduction of 27%.

The D-gluconic acid content increased to a maximum in the finishedproduct following the action of glucose oxidase/catalase on glucose. Inthis way nearly all the starting apple juice glucose was converted intogluconic acid in the apple juice product according to the presentinvention. In the 3 batch experiments, it was shown that the gluconicacid in the packaged juice product is present at a mean of 21.1 g/lcompared to below detection limits in the conventional juice. This term“gluconic acid” is generic and represents all the equilibrium species ofgluconic acid in an aqueous medium—e.g. lactones, gluconate salts forms,acid.

The magnesium content in the finished packaged juice product wasincreased to 1200 mg/kg (approximately equivalent to 1.2 g/l) comparedto less than the detection limit (<50 mg/kg) in theconventional/reference apple juice.

The Vitamin C content in the finished packaged juice product wasdecreased from 0.625 mg/100 g in the reference apple juice to anundetectable level (>0.5 mg/100 g).

There was no significant difference between the reference apple juiceand the finished packaged apple drink for a range of other chemicalparameters e.g. amino acids, vitamins, essential and trace elements,nutrients etc.

It should be noted that the classical industry standard of Brixmeasurement of free sugar content (sucrose, fructose, glucose) in fruitjuice is not appropriate for monitoring the production process accordingto the present invention because gluconic acid makes an almost identicalcontribution to the measurement as glucose. Further, the addition ofmagnesium salt also makes a small contribution to the Brix measurement.

Table 1 below shows analysis of sugar in the 3 production batches ofpackaged apple juice product according to the present invention, andcompares this with conventional apple juice made from the same rawmaterials. The conventional apple juice meets the requirements for fruitjuice laid down by Council Directive 2001/112/EC (Council of theEuropean Union, 2001), and is a representative example of the “starting”material to the juice product according to the present invention.

TABLE 1 Sugar analysis of apple juice product Sugar-depleted juiceproduct Untreated Parameter Units Batch 1 Batch 2 Batch 3 JuiceCalculation on Sugars Sugar free extract g/l 55.0 47.1 48.3 15.1(enzymatic) % sucrose % 0 0 0 26 Sum of sugars g/l 71.2 73.0 67.7 96.6(enzymatic) Individual Sugars Glucose g/l 1.7 2.1 1.7 13.5 Fructose g/l69.4 70.8 66.0 58.2 Sucrose g/l <6 <6 <6 24.9 Sugar Alcohol Sorbitolg/100 g 0.22 0.23 0.22 0.22

Example 2 Glycemic and Insulin Responses to Sugar Depleted Apple JuiceProduct I

Evidence for a beneficial effect of conversion of glucose to gluconatewith the aid of glucose oxidase on postprandial glycemic response tojuice is provided by comparing the reference food item of 400 ml applejuice (control) with the test product (400 ml apple juice treated inaccordance with the invention) in overweight (BMI 25-30) adult humanmales. Testing follows the FAO/World Health Organization (WHO)guidelines.

Test products are administered in randomized order. The drinks areingested within 5 min. Between the tests at least 2 days are interposed.At each visit the capillary blood glucose is measured 9 times over 2hours by a Haemacue meter. Capillary blood collections and glucosemeasurements are performed during the two hour interval as therecommended technique to reduce the measurement errors. Blood is drawntwice at baseline and then at times 0 (start of drink), 15 min, 30 min,45 min, 60 min, 90 min and 120 min.

Venous blood is drawn at baseline, times 0, 15, 30, 45, 60, 90 and 120min for plasma insulin determination. The incremental AUCs obtained foreach test meal are used for comparing the two meals. The 15 incrementalAUC obtained for each test meal are used for comparing the two meals.AUC of glycaemic response to treated juice is expressed as percentage ofAUC of the response to untreated juice.

Example 3 Glycemic and Insulin Responses to Sugar Depleted Apple JuiceProduct II Introduction

This study was designed to assess postprandial glycemic and insulinresponses to oral ingestion of a sugar depleted apple juice product ofthe invention and to directly compare those responses to those of theuntreated apple juice. According to the European Food Safety Authority(EFSA) the reduction of post-prandial blood glucose responses may beconsidered a beneficial physiological effect (e.g. for subjects withimpaired glucose tolerance) as long as insulin responses are notdisproportionally increased and low postprandial glycemia is givenpriority in food choice according to the Food and AgricultureOrganization of the United Nations and the World Health Organization.

Subjects and Methods Design

This mono-center, double-blind, randomized, placebo-controlled,cross-over study was conducted in 30 male volunteers aged 18 yearspresenting with a diagnosed impaired fasting plasma glucose (IFG) of5.6-6.9 mmol/L.

The study received approval from an independent ethics committee (TheEthical Committee of the Medical Council of Schleswig-Holstein, BadSegeberg, Germany) and was conducted in line with the principles of thecurrent version (2013) of the Declaration of Helsinki (WMA Declarationof Helsinki—Ethical Principles for Medical Research Involving HumanSubjects adopted by the 18th WMA (World Medical Association) GeneralAssembly, Helsinki, Finland, June 1964, and amended for the last time bythe 64th WMA General Assembly, Fortaleza, Brazil, October 2013, therecommendations for Good Clinical Practice (ICH E6), and in accordancewith European and National regulatory requirements.

Subjects

For enrollment the following inclusion criteria had to be fulfilled: Menaged 18 years diagnosis of IFG, signed Informed Consent Form. Exclusioncriteria were: Current enrollment in another clinical study, enrollmentin another clinical study within the last 4 weeks before inclusion,hypersensitivity, allergy or idiosyncratic reaction to apple, applejuice or other apple containing food, acute or chronic infections, renalinsufficiency, gastrointestinal illness, history of gastrointestinalsurgery, known fructose intolerance, overt diabetes mellitus, endocrinedisorders, any disease or condition which might compromise significantlythe hematopoietic, renal, endocrine, pulmonary, hepatic, cardiovascular,immunological, central nervous, dermatological or any other body systemwith the exception of the conditions defined by the inclusion criteria,history of hepatitis B and C, history of HIV infection, history ofcoagulation disorders or pharmaceutical anti-coagulation (with theexception of acetylsalicylic acid), regular medical treatment includingOTC, which may have impact on the study aims (e.g. antidiabetic drugs,laxatives etc.), major cognitive or psychiatric disorders, subjects whoare scheduled to undergo hospitalization during the study period, eatingdisorders (e.g. anorexia, bulimia) or special diets (e.g. vegan,vegetarian), present drug or alcohol abuse, legal incapacity.

The volunteers were free to withdraw from the study at any time withoutprejudice to their continued care. Specific reasons for discontinuingthe study were defined as: Safety reasons as judged by the Investigator,development of specific exclusion criteria during the study, which haveimpact on subject's safety, incorrect enrollment or randomization of thesubject, subject's wish to withdraw prematurely from the study, severenon-compliance to protocol as judged by the Investigator. Individualswithdrawing or discontinuing prematurely before finishing all studyvisits were supposed to be replaced, in order to have a complete set of30 subjects having completed all study visits with exploitable resultsof the primary and secondary parameters.

Random Sequence Generation and Allocation Concealment

Volunteers were randomly assigned to either Verum (sugar-depleted applejuice product of the invention) or Control product (untreated applejuice). In order to avoid selection bias, the randomization scheme wasgenerated by data managers in line with Cochrane guidelines. Therandomization list was kept confidential at the premises of Nofima ASand remained confidential with the exception of those involved inproduct production and statistical managers (after the first part ofdata locking was performed).

Test Products and Blinding of Participants and Personnel (Table 1)

Control juice. Non-pasteurized conventional apple juice was purchasedfrom Askim Frukt—og Brpresseri AS, 1815 Askim, Norway. The general applejuice production process includes: dumping, washing and grinding theapples, then pectinase treatment, cold pressing and separation. Thiscontrol product contained 17 g/L free glucose and ca. 13.5 g/L glucosebound to sucrose (Table 2).

Verum. Sugar depleted apple juice was manufactured as follows. 95 litresof control apple juice was transferred to a kettle with, mixing, heatingand cooling options (Proline Touch—Mix Kipgryde, Denmark). With lid thejuice was warmed to 85° C. and held at this temperature for 5 min. Thejuice was then cooled down (running cold water in outer jacket of thekettle) until it was 24° C. Then invertase (Maxinvert L4000, DSM) wasadded (5000 U/l) to split the non-reducing disaccharide sucrose intofructose and glucose in a overnight incubation at room temperature (ca.18-21° C.). At the end of the reaction (next morning) the content ofsucrose was determined to be <0.1 g/l.

To regulate pH prior to glucose oxidase/catalase treatment calciumhydroxide and potassium hydroxide powder was added. The enzymes glucoseoxidase/catalase (as Hyderase L, Amano/Mitsubishi) were added to thejuice (3000 U/l) followed by pure oxygen to maintain a constant supplyinto the reaction tank of 3 mg/l. pH was maintained at 3.6-4.6 by batchaddition of calcium hydroxide and potassium hydroxide powders whennecessary. An incubation time of 12 hours at room temperature wassufficient to reduce almost all glucose to gluconic acid (remainingglucose<0.1 g/l; Table 2). Glucose was monitored over the incubationperiod by reflectometric based kit (Reflectoquant from Merck). Thesucrose content after invertase incubation was also be measured in thisway (0.0 g/l; Table 2). Gluconic acid was determined via enzymatic assay(R-Biopharm). All enzyme activity was terminated by shutting off theoxygen supply. The organoleptic properties were optimized by furtheraddition of calcium and/or potassium hydroxide powder. The final pH ofthe sugar depleted drink was approximately 4. The final product waspasteurised in a KTM-Troxler (Germany) pasteur and bottled hot, corked,cooled and eventually stored in a fridge.

TABLE 2 Composition of control apple juice and sugar depleted applejuice Ingredient Apple Juice g/L Untreated (Control) Sugar depleted(Verum) Glucose 17 0.1 Fructose 65.2 86.3 Sucrose 26.7 Below detectionlimit Sugar* 109 86.4 Gluconic Acid Below detection limit 36.4 Calcium0.032 1.5 Potassium 0.960 3.100

Verum and control were similar in flavour, color, texture, andappearance and identical in packaging throughout the study and coded byconsecutive numbers in order to avoid performance bias by blinding bothstudy participants and key study personnel including outcome assessors.Code-breaking systems were available in case an adverse event occurredand medical personnel needed to be aware of what the participantreceived: (verum or control product). Raw data were also blinded duringthe blind review. The code was broken after the database was locked.

Each study participant consumed 500 mL test product at the morning ofthe interventional day (Visit 1 and 2). The 500 mL bottle content wasshaken well before opening and had to be ingested within 5 minutes.

The test products were provided by Nofima AS, Norway in a brown glassbottles and delivered to Kiel by courier service.The investigating siteensured that the study products were stored safely and properlyaccording to the instructions given by Nofima AS and kept in a securedlocation to which only the investigator and designated study staff hadaccess. The test products could be stored at room temperature with ashelf-life of two years after production. The shipment and dispensing ofstudy products was recorded in a product accountability log. Monitoringof product accountability was performed by the quality manager after thevisits and at the end of the trial.

Procedure/Conduct

Screening visit (V0). Prior to the inclusion procedure (for theassessment of eligibility of the subject), the subject was informed indetail by written information as well as verbally by the investigatorabout the study and was given the opportunity to ask the Investigatorany questions. After signing and dating the Informed Consent Form byboth the subject and the investigator subject's identity was verified,the subject's demographics and ethnics were documented, medical historywas assessed, concomitant medication and alimentary supplements, smokingand alcohol use were documented, fasting for at least 12 hours wasascertained, vital signs (blood pressure, pulse) and anthropometric data(body weight, body height) were assessed, inclusion and exclusioncriteria were assessed and blood samples for confirmation of theinclusion parameter IFG (impaired fasting glucose) were taken. Blood wasdrawn from the median cubital vein using a 21 G butterfly needle.Subjects were requested to appear the next visit after an overnightfasting of at least 12 hours and provided with a subject diary for dailyreply regarding adverse event and medication and a questionnaire (EPICFFQ) for recalling the food frequency with regard to the last 12 month,which had to be completed until the randomization visit.

Impaired fasting glucose (5.6-6.9 mmol/L resp. 100-125 mg/dL) wasconfirmed by two timely independent measures subjects are eligible forinclusion (one from the data base and one from V0).

Randomization and interventional visit 1 (V1). At V1 the study subjectswere randomized and the first intervention was performed. This visitfollowed V0 within four weeks. Adverse events happening since V0 weredocumented. If the eligibility of the subject was confirmed by theinvestigator the subject was randomized. Fasting for 12 hour prior tothis visit was checked and an intravenous catheter (Vasofix® Braunüle®,Braun Melsungen, Germany) was inserted into a forearm vein for bloodwithdrawal at baseline, directly before (time point 0) and 15, 30, 45,60, 90 and 120 minutes after starting the ingestion of the test product.From all samples plasma insulin was measured. From the blood samplestaken at baseline and 120 minutes after consumption of the test productsafety parameters were determined (Na, K, Ca, Mg, AST, ALT, γGT, CHE,AP, LDH, CK, bilirubin, creatinine, urea-N, uric acid, complete bloodcount, cholesterol, HDL-C, LDL-C, triglycerides, CRP). Capillary bloodwas taken from the finger pad using a HemoCue® Safety Lancet at baseline(twice) and once directly before (time point 0) and 15, 30, 45, 60, 90and 120 minutes after ingestion of the test product.

Arterial blood pressure, pulse and waist was assessed before and 120minutes after ingestion. Subjects completed questionnaires ongastrointestinal symptoms (Gastrointestinal Symptom Rating Scale (GSRS))directly before ingestion (time point 0) with regard to the last threedays before the visit day (V1) and with regard to the last hour beforestarting ingestion. The GSRS was also assessed 60 and 120 minutes afteringestion with respect to the last hours, each. Stool frequency andstool form was assessed directly before ingestion (time point 0) withregard to the last three days before the visit day (V1) and with regardto the last two hours before starting ingestion and also 120 minutesafter ingestion with respect to the last two hours. Satiety, hunger,fullness and prospective food consumption were monitored before (timepoint 0) and 30, 60, 90 and 120 minutes after ingestion usingquestionnaires.

Subjects were allowed to walk around, sit or lay down, but asked toabstain from eating or drinking or exercising during the test phase. Thesubjects were surveyed during the whole observation period at the testday and adverse events were monitored.

They were provided with a diary for daily assessment of adverse eventsand medication. GSRS, stool frequency and stool form were assessedduring the three day lasting observation period starting with ingestionof the test drink at visit day V1 and two subsequent days.

Interventional visit 2 (V2): This visit was scheduled on the seventh dayafter V1 at the earliest. Subjects were requested to return theirdiaries and questionnaires. Adverse events happening since V1 weredocumented. Fasting for 12 hour prior to this visit was checked and thetest was conducted as described for V1. Again they were provided with adiary for daily assessment of adverse events and medication. GSRS, stoolfrequency and stool form were assessed during the three day lastingobservation period starting with ingestion of the test drink at visitday V1 and two subsequent days. Subjects received a stamped envelope andwere requested to send back their diaries and filled questionnaires.

Assessments/Parameters

Primary target parameter. The incremental area under the curve (iAUC₁₂₀)of the capillary blood glucose levels from baseline to 120 min afteringestion of the test drinks (according to FAO/WHO) was defined asprimary parameter. Although capillary and venous blood glucose valueshave been shown to be highly correlated, capillary blood samples isregarded preferable to venous blood samples for reliable GI testing.Glucose was determined using a Hemocue 201 analyzer, Hemocue AB,Ängelholm, Sweden, which had been tested for glycemic index.

Secondary target parameter. The incremental AUC (iAUC₁₂₀) of the plasmainsulin levels from baseline to 120 min after ingestion of the testdrinks was defined as secondary parameter. Insulin was determined usingELISA (LIASION® Insulin, Diasorin S.p.A, Saluggia, Italy).

Exploratory parameters: The incremental AUC (iAUC₆₀) of glucose andinsulin levels from baseline to 120 min after ingestion of the testdrinks, the postprandial glucose peak G_(max), the amplitude betweenbaseline and G_(max) (G_(max)−G_(baseline)) and the maximal amplitude ofglucose excursions (G_(max)−G_(min)) were calculated for furthercharacterization of postprandial glucose response according toBrand-Miller (Brand-Miller et al, 2009, Am J Clin Nutr, 89: 97-105).Proportional reduction in glycaemic load (Liu, 2000) was calculated by100-100 (iAUC_(120verum)×CH_(verum))/(iAUC_(120control)×CH_(control)),whereby CH was carbohydrate (sugar) content of verum and control,respectively. Postprandial insulin sensitivity was expressed byISI=2/[AUC insulin×AUC glucose+1] according to Belfiore (Belfiore F, etal., 1998, Mol Genet Metab; 63: 134-141; and Belfiore F., 2000, DiabCare; 23:1595). Satiety, hunger, fullness and prospective food uptakewere assessed before and 30, 60, 90 and 120 min after ingestion of testdrinks according to established questionnaires.

Plasma sodium, potassium, calcium, magnesium, AST, ALT, γGT,cholinesterase, alkaline phosphatase, LDH, CK, bilirubin, creatinine,urea-N, uric acid, cholesterol, LDL-C, HDL-C, triglycerides, CRP,complete blood count, blood pressure and pulse were measured before and120 min after ingestion of test drinks.

Three days before and 3 days beginning with ingestion of the test drinksas well as 1 h before and the first and second hour after its ingestionthe gastrointestinal symptoms using the GSRS, stool frequency and stoolform using Bristol Stool Form Scale were assessed. Bristol Stool FormScale was transformed by from 1, 2, 3, 4, 5, 6 and 7 to +3, +2, +1, 0,−1, −2 and −3 for expressing deviation from normal.

Adverse events were monitored throughout the total study period andtheir severity grade (mild, moderate, severe), relationship to the studyproducts (suspected/not suspected), duration (start and end dates or ifcontinuing at final examination), the action taken and its potentialcategorisation as serious adverse event (SAE) were documented.

Statistical Analysis

Estimation of sample size: Since iAUC data were not available fromtesting apple juice, sample size estimation was based on data reportedby Johnston et al., 2003, Am J Clin Nutr; 78: 728-33. After a 25 gglucose load they found an iAUC=55.6±20.4 (mean±SD) of glycemicresponse. The glucose content of 500 mL apple juice was expected onlyabout 7 (8.5) g, the content of fructose (29.1 (33) g, GI=19%) andsucrose (12.5 (13.5) g, GI=68%) summing up to an expected glycemicresponse (7+0.19×29.1+0.68×12.5=21.03)(8.5+0.19×33+0.68×13.5=24.0) ofsimilar magnitude. We further assumed a reduction in glycemic responseby at least 30% by enzymatic treatment of the juice. Assuming areduction of iAUC by 16.7 and a SD=23.0 of the change and a power of0.95, a sample size=27 was calculated for paired t-test. Taking theweaknesses of assumptions into account a sample size of n=30 was definedfor the trial.

Preventing bias: In order to meet the recommendations of the CochraneCollaboration for preventing detection bias blinding of outcomeassessment was ensured by a blind review of raw data and by un-blindingonly after data base was locked, and by conducting statistical analysisin compliance with the statistical analysis plan. In order to avoidattrition bias, distribution of the missing data across interventiongroups and the magnitude compared to the effect size were assessed, andmissing data were replaced by the Last Observation Carried Forward(LOCF) method using the last post-baseline value for one subject at theprevious time. Reporting bias by selective outcome reporting wasprevented by the availability of the study protocol andpre-specification of (primary and secondary) outcomes and by adhering tothese specifications.

Definition of sets to be analysed: The Intention-To-Treat (ITT)collective was defined to comprise all subjects randomized and havingtaken at least one dose of the test products (intervention 1 at V1). ThePer-protocol (PP) Set comprised all subjects randomized, who have nomajor protocol deviation.

Tests: The baseline and demographic characteristics of the two groupswith different order of intervention (verum-control versuscontrol-verum) were compared using Student t or Mann-Whitney test asappropriate depending on distribution of data. Verum and control werecompared by repeated measures ANOVA, in order to take cross-over designand potential effects by the order of intervention into account. Thesignificance level of the primary and secondary parameters was adjustedto multiple testing according to Bonferroni-Holm.

Results Subject Characteristics

Distribution of volunteers through the study: N=51 subjects having hadIFG in previous studies at the study site were screened for meetinginclusion criteria and for exclusion criteria. In N=19 IFG was notverified and in N=1 individual an allergy was reported which was notreported before. Thus N=20 subjects were excluded at screening and N=31were enrolled. Between screening visit (V0) and randomization (V1) anerysipela occurred in N=1 individual. There were no drop-outs and nomajor deviations from study protocol. Thus the per protocol population(PP) was identical with intent-to-treat population (ITT) and N=30individuals, who were supposed to complete all study visits according tothe study protocol with exploitable results of the primary and secondaryparameters (PP), actually completed the study.

Population characteristics at baseline (Table 3): The total population(ITT and PP) showed features of the metabolic syndrome. The baselinecharacteristics in the group with the order verum-control (VC) did notdiffer from those in the group with the order control-verum (CV).

TABLE 3 Population characteristics at baseline (Mean ± SEM) t-Test/Mann- Total Group Group Order Group Order Whitney (n = 30) VC (n = 15)CV (n = 15) Test* Age [years] 68.0 ± 2.4 68.7 ± 1.4 67.4 ± 1.9 p = 0.602Body Height 178.0 ± 1.8  177.9 ± 2.0  178.1 ± 1.5  p = 0.911 [m] BodyWeight 100.2 ± 4.2  98.3 ± 3.3 102.0 ± 5.1  p = 0.542 [kg] BMI [kg/m²]31.6 ± 1.2 31.1 ± 1.0 32.1 ± 1.5 p = 0.581 Waist [cm] 110.9 ± 2.3  110.3± 2.7  111.8 ± 3.7  p = 0..699 Syst. Blood 131.7 ± 3.9  129.3 ± 3.9 134.0 ± 3.8  p = 0.403 Pressure [mmHg] Diastol. Blood 81.8 ± 1.8 81.0 ±2.0 82.7 ± 1.6 p = 0.524 Pressure [mmHg] Fasting Plasma 6.04 ± 0.1  6.0± 0.1 6.08 ± 0.1 * p = 0.467 Glucose [mmol/L] Fasting Plasma 166.8 ±17.9 163.6 ± 22.0 169.9 ± 28.9 p = 0.864 Triglycerides [mg/dL] FastingPlasma 49.3 ± 1.8 49.5 ± 2.4 49.2 ± 2.7 p = 0.927 HDL-C [mg/dL]

Characteristics of groups whereby those of the group with the orderverum-control (VC) were compared with those of the group with the ordercontrol-verum (CV) by Student t test in case of normal distribution ofdata and by *Mann-Whitney test, if data were not normally distributed.

Composition of Test Products (Table 2)

By enzymatic treatment glucose and sucrose were mostly removed fromapple juice by enzymatic treatment, whereas fructose increased aftercleavage of sucrose by invertase. The sugar content in g/L was reducedby 21%. The pH-value was similar between verum and control afteraddition of potassium and calcium hydroxides to the enzymaticallytreated juice. Potassium and calcium content accordingly differedbetween verum and control.

Postprandial Glycemia (FIG. 2, Table 4)

The curves of capillary blood glucose levels after ingestion of the testdrinks differed considerably between verum and control (FIG. 2B). TheiAUC₁₂₀ of glucose (primary parameter) differed significantly betweenverum and control. Similar differences were seen for iAUC₆₀, glucosemaxima, the postprandial increase from baseline and the maximal glucoseexcursion (Table 4). The order of intervention had no impact indicatingthat there were no significant carry-over effects (Table 4). Byenzymatic treatment of apple juice glycemic response to its oralingestion was significantly reduced by 68% resulting in a reduction ofglycemic load by 74.9%.

TABLE 4 Glycemic and insulin response to apple juice without (control)and with enzymatic treatment (verum) Carry- verum Control Over Parameter(N = 30) (N = 30) V versus C p Effect p iAUC₁₂₀ 63.6 ± 12.3 198.0 ±12.3  <0.001 0.806 Glucose [min × mmol/L] iAUC₆₀ 29.7 ± 5.3  108.0 ±5.3  <0.001 0.945 Glucose [min × mmol/L] Gmax 6.97 ± 0.21 8.77 ± 0.21<0.001 0.876 [mmol/L] Gmax-G 0.984 ± 0.141 2.796 ± 0.141 <0.001 0.701base [mmol/L] Gmax-Gmin 1.157 ± 0.141 3.026 ± 0.141 <0.001 0.579[mmol/L] iAUC₁₂₀ 2045 ± 285  3864 ± 285  <0.001 0.608 Insulin [min ×mU/L] iAUC₆₀ Insulin 739 ± 125 1603 ± 125  <0.001 0.401* [min × mU/L]ISI × 10⁶ 32.2 ± 3.83 4.36 ± 3.83 <0.001 0.471 iAUC₁₂₀ Glucose andiAUC₆₀ Glucose express the incremental area under the curve of capillaryblood glucose levels from ingestion to 120 and 60 min, respectively,after that. Gmax is the postprandial peak glucose level. Gmax-Gbaseexpress the increase of glucose level from baseline to Gmax. Gmax-Gminexpress the maximal glucose excursion. iAUC₁₂₀ Insulin and iAUC₆₀Insulin express the incremental area under the curve of venous plasmainsulin levels from ingestion to 120 and 60 min, respectively, afterthat. ISI = 2/[AUC insulin × AUC glucose + 1] after Belfiore. P wasassessed by ANOVA RM; *Normality failed

Postprandial Insulin (FIG. 2, Table 4)

The curves of venous plasma insulin levels after ingestion of the testdrinks differed considerably between verum and control (FIG. 2B). TheiAUC₁₂₀ (secondary parameter) differed significantly. By enzymatictreatment of apple juice insulin response to its oral ingestion wasreduced by 47%. Similar differences between verum and control were seenfor iAUC₆₀ (Table 4). Postprandial insulin sensitivity as assessed byISI differed, too (Table 4). The order of intervention had no impactindicating that there were no significant carry-over effects (Table 4).

Postprandial Safety Parameters

Plasma sodium, potassium, calcium, magnesium, AST, ALT, γGT,cholinesterase, alkaline phosphatase, LDH, CK, bilirubin, creatinine,urea-N, uric acid, cholesterol, LDL-C, HDL-C, CRP, complete blood count,blood pressure and pulse did not show clinically relevant changes andremained within the normal range 120 min after ingestion of test drinks.

Postprandial Satiety, Hunger, Fullness and Prospective Food Uptake

Satiety, hunger and prospective food uptake did not differ, neither inthe fasting state nor postprandially. Fullness differed in the fastingstate between verum (12.9±4.7) and control (24.7±4.7) (p=0.04; ANOVARM), but no longer in the following, postprandial assessments.

Gastrointestinal Symptoms

GSRS: Gastrointestinal symptoms, as assessed by the GastrointestinalSymptom Rating Scale (GSRS), did not differ 1 hour before ingestion ofthe drinks, neither the total score, nor any of the dimensions pain,reflux, indigestion, diarrhoea or constipation. Within the first hourafter ingestion the total score was higher (p=0.028) in case of verum(1.140±0.038) compared to control (1.053±0.038) and the indigestionscore was also higher (1.275±0.083) versus 1.117±0.083; p=0.008). Duringthe second hour after ingestion no differences between verum and controlwere seen. This held true within the 3 days period beginning withingestion of the test drinks.

Stool frequency did not differ between verum and control within the twohours before ingestion, but was higher (p=0.009) 2 hours after verum(0.567±0.123) compared to control (0.467±0.123). Accordingly stool form,as assessed by a transformed Bristol Stool Form Scale, was looser(p=0.002) after verum (−1.20±0.23) than after control (−0.67±0.23).Within the 3 days period beginning with ingestion of the test drinks nodifferences were reported, neither in stool frequency nor in stool form.In none of the volunteers diarrhoea as defined by WHO (three or moreloose stools per day) occurred.

Discussion

By enzymatic treatment of apple juice its sugar content in g/L could bereduced by 21% and glycemic and insulin response to oral ingestion wassignificantly reduced by 68% and 47%, respectively resulting in areduction of glycemic load by 74.9%.

1. A sugar-depleted fruit or vegetable juice product, wherein said juiceproduct is a fruit or vegetable juice or juice-retaining fruit orvegetable derived matter, wherein said juice product contains at leastabout 5 g/l gluconic acid and said juice product contains any two orthree, of (i) at least about 0.5 g/l Ca²⁺, (ii) at least about 1 g/l K⁺,and (iii) at least about 0.1 g/lMg²⁺.
 2. The sugar-depleted fruit orvegetable juice product of claim 1, wherein the combined massconcentration of free glucose and sucrose in the sugar-depleted juiceproduct is no more than about 20 g/l when said juice product is adjustedin volume with water to give a gluconic acid concentration of about 5g/l to about 100 g/l.
 3. The sugar-depleted fruit or vegetable juiceproduct of claim 2, wherein the combined mass concentration of freeglucose and sucrose in the sugar-depleted juice product is no more thanabout 15 g/l.
 4. The sugar-depleted fruit or vegetable juice product ofclaim 1, wherein the sugar-depleted juice product contains no more thanabout 5 g/l free glucose, preferably no more than about 1 g/l freeglucose, and no more than about 5 g/l sucrose, preferably no more thanabout 1 g/l sucrose, when said juice product is adjusted in volume withwater to give a gluconic acid concentration of about 5 g/l to about 100g/l or the specific sub-ranges disclosed below.
 5. The sugar-depletedfruit or vegetable juice product of claim 1, wherein the sugar-depletedjuice product is essentially devoid of free glucose and sucrose.
 6. Thesugar-depleted fruit or vegetable juice product of claim 1, wherein thesugar-depleted juice product contains at least 6 g/l gluconic acid. 7.The sugar-depleted fruit or vegetable juice product of claim 1, whereinthe sugar-depleted juice product contains at least about 0.5 g/l Ca²⁺,wherein the sugar-depleted juice product contains at least about 1 g/lK⁺, wherein the sugar-depleted juice product contains at least about 0.1g/l Mg²⁺, or a combination thereof.
 8. (canceled)
 9. (canceled)
 10. Thesugar-depleted fruit or vegetable juice product of claim 1, wherein thesugar-depleted juice product contains 5 to about 100 g/l gluconic acid.11. The sugar-depleted fruit or vegetable juice product of claim 10,wherein the sugar-depleted juice product contains about 0.5 to about 10g/l Ca²⁺, wherein the sugar-depleted juice product contains about 1 toabout 20 g/l K⁺, wherein the sugar-depleted juice product contains about0.1 to about 2 g/l Mg²⁺, wherein the sugar-depleted juice productcontains about 0.1 to about 2 g/l Mg²⁺, or a combination thereof. 12.(canceled)
 13. (canceled)
 14. The sugar-depleted fruit or vegetablejuice product of claim 1, wherein said sugar-depleted juice productcontains at least about 5 g/l gluconic acid and wherein said juiceproduct contains any two or three, of Ca²⁺, K⁺, and Mg²⁺ at a massconcentration which, when said juice product is adjusted in volume withwater to give a gluconic acid concentration of about 5 g/l, gives: (i) amass concentration of Ca²⁺ of about 0.5 to about 10 g/l (ii) a massconcentration of K⁺ of about 1 to about 20 g/l, (iii) a massconcentration of Mg²⁺ of about 0.1 to about 2 g/l.
 15. Thesugar-depleted fruit or vegetable juice product of claim 1, wherein thesugar-depleted juice product has a pH of equal to or greater than about3 and equal to or less than about
 5. 16. The sugar-depleted fruit orvegetable juice product of claim 1, wherein the sugar-depleted juicecontains Na⁺ at a mass concentration which, when said juice product isadjusted in volume with water to give a gluconic acid concentration ofbetween about 5 g/l and about 100 g/l, gives a mass concentration of Na⁺of no more than about 0.5 g/l.
 17. (canceled)
 18. A method for thepreparation of a sugar-depleted fruit or vegetable juice product asdefined in claim 1, said method comprising providing a fruit orvegetable juice product containing free glucose and/or sucrose and: (a)contacting said juice product with an enzyme which hydrolyses sucrose toglucose and fructose, (b) contacting the enzyme treated juice product ofstep (a) with an enzyme which converts glucose into gluconic acid, and(c) supplementing said juice product with one or more, preferably anytwo or three, of a source of Ca²⁺, a source of Mg²⁺ and a source of K⁺in an amount sufficient to give said mass concentrations of Ca²⁺, Mg²⁺and/or K⁺, respectively, wherein steps (a) and/or (b) may be performedsimultaneously with step (c) or before or after step (c); or said methodcomprising providing a sucrose-depleted fruit or vegetable juice productcontaining free glucose and: (d) contacting said juice product with anenzyme which converts glucose into gluconic acid, and (e) supplementingsaid juice product with one or more, preferably any two or three, of asource of Ca²⁺, a source of Mg²⁺ and a source of K⁺ in an amountsufficient to give said mass concentrations of Ca²⁺, Mg²⁺ and/or K⁺,respectively, wherein steps (d) and (e) may be performed simultaneouslyor separately in any order; or said method comprising providing asucrose-depleted fruit or vegetable juice product containing freeglucose and said mass concentrations of any two or three of Ca²⁺, Mg²⁺and/or K⁺, respectively, and: (f) contacting said juice with an enzymewhich converts glucose into gluconic acid; or said method comprisingproviding a free glucose-depleted fruit or vegetable juice product,wherein said juice product contains sucrose and at least about 5 g/lgluconic acid and: (g) contacting said juice product with an enzymewhich hydrolyses sucrose to glucose and fructose, and fh) supplementingsaid juice product with one or more, preferably any two or three, of asource of Ca²⁺, a source of Mg²⁺ and a source of K⁺ in an amountsufficient to give said mass concentrations of Ca²⁺, Mg²⁺ and/or K⁺,respectively, wherein steps (g) and (h) may be performed simultaneouslyor separately in any order; or said method comprising providing a freeglucose-depleted fruit or vegetable juice product, wherein said juiceproduct contains sucrose, at least about 5 g/l gluconic acid and saidmass concentrations of any two or three of Ca²⁺, Mg²⁺ and/or K⁺,respectively and: (i) contacting said juice product with an enzyme whichhydrolyses sucrose to glucose and fructose; or said method comprisingproviding a free glucose and optionally sucrose depleted fruit orvegetable juice product, wherein said juice product contains at leastabout 5 g/l gluconic acid and: (j) supplementing said juice product withone or more, preferably any two or three, of a source of Ca²⁺, a sourceof Mg²⁺ and a source of K⁺ in an amount sufficient to give said massconcentrations of Ca²⁺, Mg²⁺ and/or K⁺, respectively; or said methodcomprising providing a fruit or vegetable juice product containing freeglucose and/or sucrose and said mass concentrations of any two or threeof Ca²⁺, Mg²⁺ and/or K⁺, respectively, and: (k) contacting said juiceproduct with an enzyme which hydrolyses sucrose to glucose and fructose,and (l) contacting the enzyme treated juice of step (k) with an enzymewhich converts glucose into gluconic acid. wherein steps (k) and (l) maybe performed simultaneously or separately; or said method comprisingproviding a fruit or vegetable juice product containing free glucose andoptionally sucrose, and: (m) contacting said juice product with anenzyme which converts glucose into gluconic acid, and (n) supplementingsaid juice product with one or more, preferably any two or three, of asource of Ca²⁺, a source of Mg²⁺ and a source of K⁺ in an amountsufficient to give said mass concentrations of any two or three of Ca²⁺,Mg²⁺ and/or K⁺, respectively, wherein steps (m) and (n) may be performedsimultaneously or separately in any order; said method comprisingproviding a fruit or vegetable juice product containing free glucose andoptionally sucrose and said mass concentrations of any two or three ofCa²⁺, Mg²⁺ and/or K⁺, respectively, and: (o) contacting said juiceproduct with an enzyme which converts glucose into gluconic acid. 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. (canceled)
 25. (canceled)
 26. (canceled)
 27. The method of claim 18,wherein oxygen is supplied during one of more steps, preferably in theform of pure O₂.
 28. The method of claim 18, wherein said enzyme whichconverts glucose into gluconic acid is a glucose oxidase.
 29. The methodof claim 28, wherein the step of contacting the juice product with theglucose oxidase takes place in the presence of a hydrogen peroxidedegrading enzyme, preferably catalase.
 30. The method of claim 28wherein the step of contacting the juice product with the glucoseoxidase takes place at about pH 3-6, preferably about 3.4-5, about3.6-4.8, about 3.6-4.6, or about 3.8-4.4.
 31. The method of claim 18,wherein said source of Ca²⁺, Mg²⁺ and/or K⁺, are selected from salts,preferably halide salts, including fluoride, chloride, bromide, iodidesalts; organic salts, including acetate, citrate, glutamate; oxides;hydroxides; sulphates; phosphates; nitrites; nitrates and carbonates,preferably an oxide and/or a hydroxide.
 32. The method of claim 30,wherein said pH is controlled by one or more of the sources of Ca²⁺,Mg²⁺ and/or K⁺ defined in claim 31, preferably an oxide and/or ahydroxide.
 33. (canceled)
 34. A method for the preparation of foodproducts, preferably purees, sauces and toppings, jams, jellies, fruitbutters and spreads, dessert products, ice cream, juice based candiesjuice based gelatins, mixed juice products, smoothies, yoghurts,diabetic, low carbohydrate and low calorie products, dietary supplementscontaining juice, and wines and ciders, said method comprising the useof a sugar-depleted juice product of claim 1 as an ingredient in saidfood products.
 35. A method of assisting in maintaining the health andwell-being of a subject or for maintaining or promoting health andwell-being in a subject, said method comprising the subject consuming asugar-depleted juice product of claim
 1. 36. A method for the treatmentor prevention of a disease or condition associated with theover-consumption of glucose and/or sucrose and/or inappropriatemetabolism of glucose, said method comprising administering asugar-depleted juice product of claim 1 to a subject on acalorie-controlled diet.
 37. The method of claim 36, wherein saiddisease or condition associated with the over-consumption of glucoseand/or sucrose and/or inappropriate metabolism of glucose is selectedfrom metabolic syndrome, diabetes mellitus type II, obesity, abdominalobesity, dyslipidaemia, insulin resistance, hyperinsulinemia, impairedglucose metabolism, hypertension, liver steatosis, steatohepatitis,hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDLlevels, pancreatitis, neurodegenerative disease, retinopathy,nephropathy and neuropathy.
 38. A method for increasing the absorptionand retention of dietary minerals or the mineralisation of bone, saidmethod comprising administering a sugar-depleted juice of claim 1 to asubject.
 39. The method of claim 38, wherein said method treats orprevents perturbations, caused by insufficient absorbance or retentionof dietary minerals, in blood coagulation, energy-yielding metabolism,muscle function, neurotransmission, digestive enzyme function, celldivision and differentiation, development and maintenance of bones andteeth, blood pressure, the reduction of tiredness and fatigue,electrolyte balance, protein synthesis, psychological function,spermatogenesis, maintenance of hair and nails, immune system function,thyroid function, protection of DNA, proteins and lipids from oxidativedamage, DNA synthesis, carbohydrate and macronutrient metabolism,cognitive function, fertility and reproduction, maintenance of serumtestosterone concentrations, vitamin A metabolism, formation of redblood cells and haemoglobin, oxygen transport, and cognitive developmentof children.
 40. The method of claim 38, wherein said method treats orprevents bone loss disorders, preferably osteoporosis and arthritis.